Oligonucleotide Paints

ABSTRACT

Novel methods for making high resolution oligonucleotide paints are provided. Novel, high resolution oligonucleotide paints are also provided.

RELATED APPLICATIONS

This application is a continuation application which claims priority toU.S. patent application Ser. No. 17/389,257, filed on Jul. 29, 2021,which is a continuation application which claims priority to U.S. patentapplication Ser. No. 15/726,870, filed on Oct. 6, 2017, which is acontinuation application which claims priority to U.S. patentapplication Ser. No. 12/780,446, filed on May 14, 2010, which claims thebenefit of U.S. provisional patent application Nos. 61/183,247, filedJun. 2, 2009, and 61/228,931, filed Dec. 22, 2009, each of which arehereby incorporated by reference in their entireties.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under the NationalInstitutes of Health grant number GM085169-01A1. The Government hascertain rights in the invention.

FIELD

Embodiments of the present invention relate in general methods formaking and using oligonucleotide paints for chromosome analysis methods.

BACKGROUND

Cytogeneticists have been working hand-in-hand with geneticists andmolecular geneticists to clarify the processes of inheritance and geneexpression ever since the synergy of August Weissman's chromosome theoryof inheritance, as interpreted by Boveri and Sutton in 1902, withMendel's theory of inheritance, as brought forth by Morgan, Sturtevant,Muller, and Bridges in their landmark 1915 publication, The Mechanism ofMendelian Heredity. This synergy, however, has become technologicallyunbalanced, as the tools for dissecting gene expression outstrip thosewith which cytogeneticists tease apart the arrangement of chromosomeswithin the nucleus or their behavior as they or are inherited fromcell-to-cell or generation-to-generation.

Chromosome arrangement and behavior cannot be extracted, purified orcaptured. Chromosomes have no unit structure that can be isolated andcrystallized, and they produce no product or enzymatic activity that canbe assayed in a test tube. Instead, researchers must study chromosomearrangement and behavior in situ, visualizing them with cytologicaltools or via genetic manipulation. Constrained as well as guided bythese requirements, remarkable technologies have nonetheless beendeveloped. Cytology-grade microscopes, electron microscopes, chromosomestains, and in situ hybridization protocols have all greatly advancedthe ability of scientists to study how chromosome organization impactsgene expression and development. For example, the use of fluorescent insitu hybridization (FISH) to reveal the co-localization of the Myc andIgh genes in transcription factories, provides a plausible explanationfor the frequency with which these two genes, lying on differentchromosomes, become fused through translocations associated withplasmacytoma and Burkitt lymphoma (Osborne et al. (2007) PLoS Biol.5(8):e192). Studies such as this can only be carried out in situ,highlighting the need for cytological technologies. Most recently, thetechnology of chromosome conformation capture (Ohlsson et al. (2007)Curr. Opin. Cell Biol. 19(3):321) has fused molecular biological toolsand cytological tools to capture and clone chromosomal regions that comeinto contact, generating tremendous excitement among geneticists andcytogeneticists. Although indirect, genetic approaches have elucidatedthe manner by which chromosomes are transmitted through mitosis andmeiosis into subsequent cellular and organismal generations and, throughthe use of translocations and chromosomal rearrangements, demonstratedhow chromosome positioning and interchromosomal interactions canprofoundly affect gene expression (Wu et al. (1999) Curr. Opin. Gen.Dev. 9:237; Duncan (2002) Ann. Rev. Genet. 36:521; Grant-Downton, et al.(2004) Trends Genet. 20:188; McKee (2004) Biochim Biophys Acta 1677:165;Zickler (2006) Chromosoma 115:158).

Still, scientists remain tremendously limited in the ability tounderstand the relationship between chromosome arrangement and geneexpression. Foremost among these needs are technologies that will permitthe visualization of chromosome arrangement, single nucleus by singlenucleus, a need that grows as evidence accumulates steadily for theroles that chromosome positioning and interchromosomal interactions playin the regulation of genes and development in humans and other mammals,Drosophila, plants, nematodes, fungi and, essentially, every species.

SUMMARY

Chromosome paints are detectable markers that label chromosomes alongtheir entire length, permitting physicians and researchers to identifychromosomes and decipher chromosome rearrangements. However,commercially available paints are expensive for routine and frequentuse, ranging between $100 to $4,000 or more per whole genome, per assay,with increased resolution requiring more expensive paints. As such, manyresearchers have not utilized chromosome paints for systematicgenome-wide analysis and have, instead, used the existing chromosomepaint technology sparingly.

It has been surprisingly discovered that chromosome paints havingsuperior resolution and labeling functionality can be economicallygenerated using the methods described herein. It has been discoveredthat the per assay cost of chromosome paints could be reducedapproximately 50- to 4,000-fold while increasing resolution by 100- to1,000-fold or more, thus rendering possible many diagnoses and researchprojects that would otherwise not be performed or considered due toprohibitive cost. For example, the methods and compositions describedherein can be used to produce paints for all chromosomes of the humangenome for as little as $1 to $2 per assay.

Accordingly, a first method of making a set of high resolutionoligonucleotide paints is provided. The method includes the steps ofproviding at least one solid support having a plurality of synthetic,single stranded oligonucleotide sequences attached thereto, wherein aportion of each of the plurality of synthetic, single strandedoligonucleotide sequences is complementary to a portion of a specificchromosome sequence, synthesizing a plurality of complementary strands,each of which is complementary to a synthetic, single strandedoligonucleotide sequence attached to the at least one solid support,removing the plurality of complementary strands from the at least onesolid support, amplifying the plurality of complementary strands, andlabelling the plurality of complementary strands to produce a set ofoligonucleotide paints, wherein the set oligonucleotide paints has aresolution of about two kilobases or fewer. In certain aspects, eacholigonucleotide paint has a resolution of about one kilobase or fewer or100 bases or fewer. In certain aspects, the set of oligonucleotidepaints has a resolution of between about 20 bases and about 30 bases. Incertain aspects, the length of each of the oligonucleotide sequences isabout 60 bases (e.g., about 14 bases at each of the 3′ and 5′ ends of anoligonucleotide sequence are primer sequences and about 32 basesinternal to the primer sequences are complementary to a chromosomesequence). In other aspects, each of the oligonucleotide paints has adetectable and/or retrievable label attached thereto. In certainaspects, the retrievable label further binds a moiety selected from thegroup consisting of a protein, a peptide, a DNA sequence, an RNAsequence and a carbohydrate. In other aspects, the retrievable moiety isexposed to light, heat or a chemical to activate binding of theretrievable label to a moiety selected from the group consisting of aprotein, a peptide, a DNA sequence, an RNA sequence and a carbohydrate.In certain aspects, each of the oligonucleotide paints has a detectablelabel attached thereto. In certain aspects, the detectable label is afluorescent label. In other aspects, the set of oligonucleotide paintsprovides one spectrally resolvable color, or at least 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,50, 100, 200, 300, 400, 500 or more spectrally resolvable labels and/orthe set of oligonucleotide paints provides a spectrally resolvable labelfor each chromosome and/or one or more sub-chromosomal regions of anorganism. In certain aspects, the plurality of synthetic, singlestranded oligonucleotide sequences encodes 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75%, 80%, 85%,90%, 95%, 99% or more (e.g., 100%) of a genome (e.g., a human genome) orbetween 1% and 75%, between 5% and 50%, between 5% and 25%, between 5%and 75%, between 10% and 50% or between 20% and 40% of a genome (e.g., ahuman genome). In certain aspects, at least 25 microarrays or at least100 microarrays are provided that are optionally generated and/orutilized in parallel. In still other aspects, the step of amplifyingincludes providing a plurality of primers (e.g., universal primers),each of which is complementary to a portion of a complementary strand ora portion of a single stranded oligonucleotide sequence. In yet otheraspects, at least a portion of each of the primer sequences is removableafter the amplification step. In certain aspects, the universal primerscomprise between one and 1000 different sequences or comprise at least1000 different sequences. In other aspects, a set of oligonucleotidepaints produced by the first method is provided. In still other aspects,a method of detecting a chromosome rearrangement in a biological sample(e.g., one or more of translocation, insertion, inversion, deletion,duplication, transposition, aneuploidy, polyploidy, complexrearrangement and telomere loss) including the steps of providing abiological sample, contacting the biological sample with the set ofoligonucleotide paints of the first method, detecting binding of the setof oligonucleotide paints, comparing the binding of the set ofoligonucleotide paints to a standard, and detecting a chromosomerearrangement if binding of the set of oligonucleotide paints differsfrom the standard is provided.

A second method of making a set of oligonucleotide paints is provided.The method includes the steps of providing at least one solid supporthaving a plurality of synthetic, single stranded oligonucleotidesequences attached thereto, wherein a portion of each of the pluralityof synthetic, single stranded oligonucleotide sequences is complementaryto a portion of a specific chromosome sequence and wherein each specificchromosome sequence excludes highly repetitive elements (and/or anyother genomic sequence that one wants to exclude), synthesizing aplurality of complementary strands, each of which is complementary to asynthetic, single stranded oligonucleotide sequence attached to the atleast one solid support, removing the plurality of complementary strandsfrom the at least one solid support, amplifying the plurality ofcomplementary strands, and labelling the plurality of complementarystrands to produce a set of oligonucleotide paints. In certain aspects,each specific chromosome sequence excludes repetitive elements presentin the genome as two copies, three copies or four copies (i.e., in ahaploid genome). In other aspects, the length of each of theoligonucleotide sequences is about 60 bases (e.g., about 14 bases ateach of the 3′ and 5′ ends of an oligonucleotide sequence are primersequences and about 32 bases internal to the primer sequences arecomplementary to a chromosome sequence). In certain aspects, each of theoligonucleotide paints has a retrievable label attached thereto. Incertain aspects, the retrievable label further binds a moiety selectedfrom the group consisting of a protein, a peptide, a DNA sequence, anRNA sequence and a carbohydrate. In other aspects, the retrievablemoiety is exposed to light, heat or a chemical to activate binding ofthe retrievable label to a moiety selected from the group consisting ofa protein, a peptide, a DNA sequence, an RNA sequence and acarbohydrate. In certain aspects, each of the oligonucleotide paints hasa detectable label attached thereto. In certain aspects, the detectablelabel is a fluorescent label. In other aspects, the set ofoligonucleotide paints provides one spectrally resolvable color, or atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 50, 100, 200, 300, 400, 500 or more spectrallyresolvable labels and/or the set of oligonucleotide paints provides aspectrally resolvable label for each chromosome and/or one or moresub-chromosomal regions of an organism. In certain aspects, theplurality of synthetic, single stranded oligonucleotide sequencesencodes 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 66%, 70%, 75% or more of a genome (e.g., a human genome) orbetween 1% and 75%, between 5% and 50%, between 5% and 25%, between 5%and 75%, between 10% and 50% or between 20% and 40% of a genome (e.g., ahuman genome). In certain aspects, at least 25 microarrays or at least100 microarrays are provided that are optionally generated and/orutilized in parallel. In still other aspects, the step of amplifyingincludes providing a plurality of primers (e.g., universal primers),each of which is complementary to a portion of a complementary strand ora portion of a single stranded oligonucleotide sequence. In yet otheraspects, at least a portion of each of the primer sequences is removableafter the amplification step. In certain aspects, the universal primerscomprise between one and 1000 different sequences or comprise at least1000 different sequences. In other aspects, a set of oligonucleotidepaints produced by the second method is provided. In still otheraspects, a method of detecting a chromosome rearrangement in abiological sample (e.g., one or more of translocation, insertion,inversion, deletion, duplication, transposition, aneuploidy, polyploidy,complex rearrangement and telomere loss) including the steps ofproviding a biological sample, contacting the biological sample with theset of oligonucleotide paints of the second method, detecting binding ofthe set of oligonucleotide paints, comparing the binding of the set ofoligonucleotide paints to a standard, and detecting a chromosomerearrangement if binding of the set of oligonucleotide paints differsfrom the standard is provided.

In certain exemplary embodiments, a palette of oligonucleotide paintsincluding a plurality of oligonucleotide sequences, wherein eacholigonucleotide sequence is complementary to a single type of mutationcorresponding to one of a specific set of chromosome abnormalitiesassociated with a disorder, and wherein the set comprises at least 50different types of mutations is provided. In certain aspects, the setincludes at least 100 different types of mutations, at least 1000different types of mutations, at least 10,000 different types ofmutations or more.

In certain exemplary embodiments a kit (e.g., a diagnostic kit)including the set of oligonucleotide paints of the first or secondmethod is provided. In certain aspects the kit includes instructions foruse. In other aspects, the kit is used to determine the karyotype of asample.

In certain exemplary embodiments, an article of manufacture for making aset of high resolution oligonucleotide paints is provided, including aplurality of microarrays, each microarray having a plurality ofsynthetic oligonucleotide sequences attached thereto, wherein a portionof each of the plurality of synthetic oligonucleotide sequences iscomplementary to a portion of a specific chromosome sequence, whereinthe sum of synthetic oligonucleotide that are complementary correspondsto between about 5% and 25% of a genome of interest, and wherein the setof oligonucleotide paints has a resolution of about two kilobases orfewer. In other aspects, the plurality of synthetic oligonucleotidesequences is complementary to approximately 1%, 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 66%, 70%, 75% or moreof a genome (e.g., a human genome). In still other aspects, the articleof manufacture further includes a plurality of primers (e.g., universalprimers).

In certain exemplary embodiments, a method of preparing a plurality ofhigh resolution oligonucleotide paints comprising computationallydetermining genomic spacing of a plurality of synthetic, oligonucleotidesequences, wherein each of the plurality is complementary to a portionof a specific chromosome sequence, synthesizing the plurality ofsynthetic oligonucleotide sequences, and labelling the plurality ofsynthetic oligonucleotide sequences with a detectable label to produce aplurality of oligonucleotide paints, wherein the set of oligonucleotidepaints has a resolution of about two kilobases or fewer, and whereineach of a plurality of the oligonucleotide paints is complementary to atarget nucleic acid sequence (e.g., a genomic sequence) of 40consecutive nucleotide bases or fewer is provided. In certain aspects,the plurality of the oligonucleotide paints is complementary to a targetnucleic acid sequence of 30, 20, 10 or fewer consecutive nucleotidebases. In certain aspects, the method further includes the step ofcomputationally selecting at least one detectable label to label each ofthe plurality of synthetic, oligonucleotide sequences. In other aspects,the method further includes the step of computationally determining thepresence of single nucleotide polymorphisms in a genomic sequence ofinterest to reduce synthesis of synthetic oligonucleotide sequences thatbind to repeated regions of the genomic sequence of interest.

In certain exemplary embodiments, a method of making a set of highresolution oligonucleotide paints comprising providing at least onesolid support having a plurality of synthetic, single strandedoligonucleotide sequences attached thereto, wherein a portion of each ofthe plurality of synthetic, single stranded oligonucleotide sequences iscomplementary to a portion of a specific chromosome sequence,synthesizing a plurality of complementary strands, each of which iscomplementary to a synthetic, single stranded oligonucleotide sequenceattached to the at least one solid support, removing the plurality ofcomplementary strands from the at least one solid support, amplifyingthe plurality of complementary strands, and labelling the plurality ofcomplementary strands to produce a set of high resolutionoligonucleotide paints, wherein each of a plurality of theoligonucleotide paints is complementary to a target nucleic acidsequence of 40 consecutive nucleotide bases or fewer is provided. Incertain aspects, the plurality of the oligonucleotide paints iscomplementary to a target nucleic acid sequence of 30, 20, 10 or fewerconsecutive nucleotide bases. In certain aspects, the oligonucleotidepaints can cross a cell membrane and/or a nuclear membrane. In otheraspects, the oligonucleotide paints include a detectable label (e.g., afluorescent label) and a quencher. The quencher can optionally bereleased during the step of extension. In certain aspects, the targetnucleic acid sequences are present in a multi-well (e.g., a 384-well)plate. In other aspects, hybridized oligonucleotide paints are detectedby fluorescent in situ hybridization (FISH). In certain aspects, thetarget nucleic acid sequence is genomic.

In other aspects, a method described herein further includes the step ofhybridizing the oligonucleotide paints to one or more target sequences.In still other aspects, a method described herein further includes thestep of extending the plurality of hybridized oligonucleotide paints(e.g., by primer extension). In yet other aspects, a method describedherein includes the step of washing the extended plurality of hybridizedoligonucleotide paints under stringent conditions. In other aspects, amethod described herein further includes the step of hybridizing theoligonucleotide paints to one or more target sequences in the presenceof an enzyme selected from the group consisting of one or more of aproteinase, a lipase, and a ribonuclease.

Further features and advantages of certain embodiments of the presentinvention will become more fully apparent in the following descriptionof the embodiments and drawings thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings in which:

FIG. 1 schematically depicts one method of making of chromosome paints(i.e., Oligopaints) from oligonucleotides.

FIG. 2 schematically depicts one method of making 20% of the humangenome on up to 92 arrays which, after amplification with between 5 and15 (=5+1+1+1+7) different kinds of primer pairs, will generate 664 poolsof genomic sequence. The pools can be combined in a variety of ways totarget chromosomes or sub-chromosomal regions.

FIG. 3 schematically depicts how thick banding will become a finerpattern on decondensed chromosomes.

FIG. 4 schematically depicts strategies for aliquoting. Either strategyallows for the visualization of just one or a few chromosomes at a time,as well as permitting the visualization of sub-chromosomal regions.Aliquots carrying different primer sequences can be labeled with thesame marker if there is no need to distinguish the targets byfluorescent in situ hybridization (FISH).

FIG. 5 schematically depicts one protocol to make Oligopaint probes fromchip-synthesized oligonucleotide libraries.

FIG. 6 depicts an RNAi screen for genes involved in Drosophila cells,locked nucleic acid (LNA) probes and automated scoring.

FIG. 7 schematically depicts the use of PCR primers that include aninternal dU and an internal fluor, enabling digestion of the 5′ end ofthe primers with USER™ (uracil-specific excision reagent) (New EnglandBiolabs, Ipswich, Mass.).

DETAILED DESCRIPTION

The principles of the present invention may be applied with particularadvantage in methods of tagging (i.e., painting with chromosome paints)one or more oligonucleotide sequences, e.g., chromosome regions (e.g.,sub-chromosomal regions) and/or one or more entire chromosomes. Themethods described herein create chromosome paints that have an increasedresolution over commercially available chromosome paints, which is duein part to the fact that the chromosome paints are synthesized using aspecific set of primers, which can amplify and label specific sequenceswith near absolute certainty. Thus, the chromosome paints describedherein have a theoretical resolution on the order of base pairs.

Exemplary embodiments of the present invention are directed to methodsfor generating novel chromosome paints using synthetic genomic templatesequences (e.g., genomic template sequences that have been synthesizedon arrays). The synthetic genomic template sequences can be, forexample, synthetic genomic template sequences that are generated on andsubsequently released from an array into one or more pools, or extensionproducts which are made using synthetic genomic template sequencesattached to an array as a template and then released into one or morepools by melting. The released sequences are then amplified and labeledto produce chromosome paints. By designing synthetic genomic templatesequences to be flanked by primer sequences, the primers can be usedboth to label the synthetic genomic template sequences as well as toamplify the genomic sequence. Labeling a chromosome paint can beperformed by a variety of methods including, but not limited to, usingprimers that have been pre-labeled, incorporating labels duringamplification or indirect labeling. Labels and methods of incorporatinglabels into oligonucleotide sequences are discussed further herein.

As used herein, the term “chromosome paint” refers to detectably labeledpolynucleotides that have sequences complementary to DNA sequences froma particular chromosome or sub-chromosomal region of a particularchromosome. Chromosome paints that are commercially available arederived from fluorescence activated cell sorted (FACS) and/or flowsorted chromosomes or from bacterial artificial chromosomes (BACs) oryeast artificial chromosomes (YACs). As such, chromosome paints known inthe art at the time of filing were laborious to generate and are limitedin their resolution.

As used herein, the term “Oligopaint” refers to detectably labeledpolynucleotides that have sequences complementary to an oligonucleotidesequence, e.g., a portion of a DNA sequence e.g., a particularchromosome or sub-chromosomal region of a particular chromosome.Oligopaints are generated from synthetic probes and arrays that are,optionally, computationally patterned (rather than using natural DNAsequences and/or chromosomes as a template).

Since Oligopaints are generated using nucleic acid sequences that arepresent in a pool, they are no longer spatially addressable (i.e., nolonger attached to an array). Surprisingly, however, this methodincreases resolution of the chromosome paints over those that are madeusing yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), and/or flow sorted chromosomes. In certain aspects,the Oligopaints described herein have a resolution that is, e.g., 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%,300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%,3000%, 4000%, 5000%, 6000%, 7000%, 8000%, 9000%, 10,000%, 100,000%,1,000,000%, 10,000,000%, 100,000,000% or greater than chromosome paintsthat are commercially available.

Typically, chromosome paints that are commercially available have achromosome resolution on the order of at least 6×10⁶ base pairs. TheOligopaints described herein, however, have a much higher resolutionwhen compared with paints known in the art. As used herein, the term“resolution” refers to the ability to distinguish (e.g., label) betweentwo points on a polynucleotide sequence (e.g., two points along thelength of a chromosome). As used herein, the term “high resolution”refers to the ability to detect two or more nucleic acid sequenceshaving a distance of less than 6×10⁶ base pairs apart (e.g., on achromosome). In certain aspects, two or more high resolution Oligopaintshave a resolution of about 500 kilobases apart or fewer, 400 kilobasesapart or fewer, 300 kilobases apart or fewer, 200 kilobases apart orfewer, 100 kilobases apart or fewer, 90 kilobases apart or fewer, 80kilobases apart or fewer, 70 kilobases apart or fewer, 60 kilobasesapart or fewer, 50 kilobases apart or fewer, 40 kilobases apart orfewer, 30 kilobases apart or fewer, 20 kilobases apart or fewer, 19kilobases apart or fewer, 18 kilobases apart or fewer, 17 kilobasesapart or fewer, 16 kilobases apart or fewer, 15 kilobases apart orfewer, 14 kilobases apart or fewer, 13 kilobases apart or fewer, 12kilobases apart or fewer, 11 kilobases apart or fewer, 10 kilobasesapart or fewer, 9 kilobases apart or fewer, 8 kilobases apart or fewer,7 kilobases apart or fewer, 6 kilobases apart or fewer, 5 kilobasesapart or fewer, 4 kilobases apart or fewer, 3 kilobases apart or fewer,2 kilobases apart or fewer or 1 kilobase apart or fewer. In certainaspects, two or more high resolution Oligopaints have a resolution ofabout 1900 bases apart or fewer, 1800 bases apart or fewer, 1700 basesapart or fewer, 1600 bases apart or fewer, 1500 bases apart or fewer,1400 bases apart or fewer, 1300 bases apart or fewer, 1200 bases apartor fewer, 1100 bases apart or fewer, 1000 bases apart or fewer, 900bases apart or fewer, 800 bases apart or fewer, 700 bases apart orfewer, 600 bases apart or fewer, 500 bases apart or fewer, 400 basesapart or fewer, 300 bases apart or fewer, 200 bases apart or fewer, 100bases apart or fewer, 95 bases apart or fewer, 90 bases apart or fewer,85 bases apart or fewer, 80 bases apart or fewer, 75 bases apart orfewer, 70 bases apart or fewer, 65 bases apart or fewer, 60 bases apartor fewer, 55 bases apart or fewer, 50 bases apart or fewer, 45 basesapart or fewer, 40 bases apart or fewer, 35 bases apart or fewer, 30bases apart or fewer, 25 bases apart or fewer, 20 bases apart or fewer,15 bases apart or fewer, 10 bases apart or fewer or down to theindividual base pair. In certain aspects, two or more high resolutionOligopaints have a resolution of between about 10 bases and about 2000bases, between about 10 bases and about 1000 bases, between about 10bases and about 500 bases, between about 15 bases and about 250 bases,between about 15 bases and about 100 bases, between about 20 bases andabout 50 bases, or between about 20 bases and about 30 bases.

The sensitivity of resolution of Oligopaints described herein is muchgreater than paints known in the art. As used herein, the term“sensitivity,” with respect to Oligopaints, refers to the number oftarget nucleotide bases (e.g., target genomic nucleotide bases) that arecomplementary to a particular Oligopaint, i.e., the number of targetnucleotide bases to which a particular Oligopaint can hybridize (i.e.,the smallest band size that can be detected). In certain aspects, highresolution Oligopaints have a resolution of about 1 kilobase, about 1900bases, about 1800 bases, about 1700 bases, about 1600 bases apart, about1500 bases, about 1400 bases, about 1300 bases, about 1200 bases, about1100 bases, about 1000 bases, about 900 bases, about 800 bases, about700 bases, about 600 bases, about 500 bases, about 400 bases, about 300bases, about 200 bases, about 100 bases, about 95 bases, about 90 bases,about 85 bases, about 80 bases, about 75 bases, about 70 bases, about 65bases, about 60 bases, about 55 bases, about 50 bases, about 45 bases,about 40 bases, about 35 bases, about 30 bases, about 25 bases, about 20bases, about 15 bases, about 10 bases, or about 5 bases. In certainaspects, the number of target nucleotide bases that are complementary toan Oligopaint are consecutive (e.g., consecutive genomic nucleotidebases).

In certain exemplary embodiments, Oligopaints are complementary togenomic nucleic sequences that are present in low or single copy numbers(e.g., genomic nucleic sequences that are not repetitive elements). Asused herein, the term “repetitive element” refers to a DNA sequence thatis present in many identical or similar copies in the genome. Repetitiveelements are not intended to refer to a DNA sequence that is present oneach copy of the same chromosome (e.g., a DNA sequence that is presentonly once, but is found on both copies of chromosome 11, would not beconsidered a repetitive element, and would be considered a sequence thatis present in the genome as one copy). The genome consists of threebroad sequence components: Single copy or at least very low copy numberDNA (approximately 60% of the human genome); moderately repetitiveelements (approximately 30% of the human genome); and highly repetitiveelements (approximately 10% of the human genome). For a review, seeHuman Molecular Genetics, Chapter 7 (1999), John Wiley & Sons, Inc.

In certain exemplary embodiments, small Oligopaints are provided. Asused herein, the term “small Oligopaint” refers to an Oligopaint ofbetween about 5 bases and about 100 bases long, or an Oligopaint ofabout 5 bases, about 10 bases, about 15 bases, about 20 bases, about 25bases, about 30 bases, about 35 bases, about 40 bases, about 45 bases,about 50 bases, about 55 bases, about 60 bases, about 65 bases, about 70bases, about 75 bases, about 80 bases, about 85 bases, about 90 bases,about 95 bases, or about 100 bases. Small Oligopaints can access targetsthat are not accessible to longer oligonucleotide probes. For example,in certain aspects small Oligopaints can pass into a cell, can pass intoa nucleus, and/or can hybridize with targets that are partially bound byone or more proteins, etc. Small Oligopaints are also useful forreducing background, as they can be more easily washed away than largerhybridized oligonucleotide sequences.

In certain exemplary embodiments, the length of an Oligopaint can beincreased (e.g., by primer extension) after it has been hybridized to atarget sequence, e.g., a target genomic sequence. Such an extension canincrease the binding affinity of the Oligopaint to the target sequence,allowing more stringent hybridization and/or wash conditions to be used(temperature, salt concentration, detergent concentration and the like,discussed further herein) as compared to a shorter Oligopaint whilestill allowing the use of small Oligopaints. In certain aspects, the useof stringent hybridization and/or wash conditions improves the signal tonoise ratio of an Oligopaint.

As used herein, the terms “Oligopainted” and “Oligopainted region” referto a target nucleotide sequence (e.g., a chromosome) or region of atarget nucleotide sequence (e.g., a sub-chromosomal region),respectively, that has hybridized thereto one or more Oligopaints.Oligopaints can be used to label a target nucleotide sequence, e.g.,chromosomes and sub-chromosomal regions of chromosomes during variousphases of the cell cycle including, but not limited to, interphase,preprophase, prophase, prometaphase, metaphase, anaphase, telophase andcytokinesis.

As used herein, the term “chromosome” refers to the support for thegenes carrying heredity in a living cell, including DNA, protein, RNAand other associated factors. The conventional international system foridentifying and numbering the chromosomes of the human genome is usedherein. The size of an individual chromosome may vary within amulti-chromosomal genome and from one genome to another. A chromosomecan be obtained from any species. A chromosome can be obtained from anadult subject, a juvenile subject, an infant subject, from an unbornsubject (e.g., from a fetus, e.g., via prenatal test such asamniocentesis, chorionic villus sampling, and the like or directly fromthe fetus, e.g., during a fetal surgery) from a biological sample (e.g.,a biological tissue, fluid or cells (e.g., sputum, blood, blood cells,tissue or fine needle biopsy samples, urine, cerebrospinal fluid,peritoneal fluid, and pleural fluid, or cells therefrom) or from a cellculture sample (e.g., primary cells, immortalized cells, partiallyimmortalized cells or the like). In certain exemplary embodiments, oneor more chromosomes can be obtained from one or more genera including,but not limited to, Homo, Drosophila, Caenorhabiditis, Danio, Cyprinus,Equus, Canis, Ovis, Ocorynchus, Salmo, Bos, Sus, Gallus, Solanum,Triticum, Oryza, Zea, Hordeum, Musa, Avena, Populus, Brassica, Saccharumand the like.

As used herein, the term “chromosome banding” refers to differentialstaining of chromosomes resulting in a pattern of transverse bands ofdistinguishable (e.g., differently or alternately colored) regions, thatis characteristic for the individual chromosome or chromosome region(i.e., the “banding pattern”). Conventional banding techniques includeG-banding (Giemsa stain), Q-banding (Quinacrine mustard stain),R-banding (reverse-Giemsa), and C-banding (centromere banding).

As used herein, the term “karyotype” refers to the chromosomecharacteristics of an individual cell, cell line or genome of a givenspecies, as defined by both the number and morphology of thechromosomes. Karyotype can refer to a variety of chromosomalrearrangements including, but not limited to, translocations,insertional translocations, inversions, deletions, duplications,transpositions, anueploidies, complex rearrangements, telomere loss andthe like. Typically, the karyotype is presented as a systematized arrayof prophase or metaphase (or otherwise condensed) chromosomes from aphotomicrograph or computer-generated image. Interphase chromosomes mayalso be examined.

As used herein, the terms “chromosomal aberration” or “chromosomeabnormality” refer to a deviation between the structure of the subjectchromosome or karyotype and a normal (i.e., non-aberrant) homologouschromosome or karyotype. The deviation may be of a single base pair orof many base pairs. The terms “normal” or “non-aberrant,” when referringto chromosomes or karyotypes, refer to the karyotype or banding patternfound in healthy individuals of a particular species and gender.Chromosome abnormalities can be numerical or structural in nature, andinclude, but are not limited to, aneuploidy, polyploidy, inversion,translocation, deletion, duplication and the like. Chromosomeabnormalities may be correlated with the presence of a pathologicalcondition or with a predisposition to developing a pathologicalcondition. Chromosome aberrations and/or abnormalities can also refer tochanges that are not associated with a disease, disorder and/or aphenotypic change. Such aberrations and/or abnormalities can be rare orpresent at a low frequency (e.g., a few percent of the population (e.g.,polymorphic)).

Disorders associated with one or more chromosome abnormalities include,but are not limited to: autosomal abnormalities (e.g., trisomies (Downsyndrome (chromosome 21), Edwards syndrome (chromosome 18), Patausyndrome (chromosome 13), trisomy 9, Warkany syndrome (chromosome 8),trisomy 22/cat eye syndrome, trisomy 16); monosomies and/or deletions(Wolf-Hirschhorn syndrome (chromosome 4), Cri du chat/Chromosome 5qdeletion syndrome (chromosome 5), Williams syndrome (chromosome 7),Jacobsen syndrome (chromosome 11), Miller-Dieker syndrome/Smith-Magenissyndrome (chromosome 17), Di George's syndrome (chromosome 22), genomicimprinting (Angelman syndrome/Prader-Willi syndrome (chromosome 15)));X/Y-linked abnormalities (e.g., monosomies (Turner syndrome (XO),trisomy or tetrasomy and/or other karyotypes or mosaics (Klinefelter'ssyndrome (47 (XXY)), 48 (XXYY), 48 (XXXY), 49 (XXXYY), 49 (XXXXY),Triple X syndrome (47 (XXX)), 48 (XXXX), 49 (XXXXX), 47 (XYY), 48(XYYY), 49 (XYYYY), 46 (XX/XY)); translocations (e.g., leukemia orlymphoma (e.g., lymphoid (e.g., Burkitt's lymphoma t(8 MYC; 14 IGH),follicular lymphoma t(14 IGH; 18 BCL2), mantle cell lymphoma/multiplemyeloma t(11 CCND1; 14 IGH), anaplastic large cell lymphoma t(2 ALK; 5NPM1), acute lymphoblastic leukemia) or myeloid (e.g., Philadelphiachromosome t(9 ABL; 22 BCR), acute myeloblastic leukemia with maturationt(8 RUNX1T1;21 RUNX1), acute promyelocytic leukemia t(15 PML,17 RARA),acute megakaryoblastic leukemia t(1 RBM15;22 MKL1))) or other (e.g.,Ewing's sarcoma t(11 FLI1; 22 EWS), synovial sarcoma t(x SYT;18 SSX),dermatofibrosarcoma protuberans t(17 COL1A1; 22 PDGFB), myxoidliposarcoma t(12 DDIT3; 16 FUS), desmoplastic small round cell tumort(11 WT1; 22 EWS), alveolar rhabdomyosarcoma t(2 PAX3; 13 FOXO1) t (1PAX7; 13 FOXO1))); gonadal dysgenesis (e.g., mixed gonadal dysgenesis,XX gonadal dysgenesis); and other abnormalities (e.g., fragile Xsyndrome, uniparental disomy). Disorders associated with one or morechromosome abnormalities also include, but are not limited to,Beckwith-Wiedmann syndrome, branchio-oto-renal syndrome, Cri-du-Chatsyndrome, De Lange syndrome, holoprosencephaly, Rubinstein-Taybisyndrome and WAGR syndrome.

Disorders associated with one or more chromosome abnormalities alsoinclude cellular proliferative disorders (e.g., cancer). As used herein,the term “cellular proliferative disorder” includes disorderscharacterized by undesirable or inappropriate proliferation of one ormore subset(s) of cells in a multicellular organism. The term “cancer”refers to various types of malignant neoplasms, most of which can invadesurrounding tissues, and may metastasize to different sites (see, forexample, PDR Medical Dictionary 1st edition, 1995). The terms “neoplasm”and “tumor” refer to an abnormal tissue that grows by cellularproliferation more rapidly than normal and continues to grow after thestimuli that initiated proliferation is removed (see, for example, PDRMedical Dictionary 1st edition, 1995). Such abnormal tissue showspartial or complete lack of structural organization and functionalcoordination with the normal tissue which may be either benign (i.e.,benign tumor) or malignant (i.e., malignant tumor).

Disorders associated with one or more chromosome abnormalities alsoinclude brain disorders including, but not limited to, acoustic neuroma,acquired brain injury, Alzheimer's disease, amyotrophic lateraldiseases, aneurism, aphasia, arteriovenous malformation, attentiondeficit hyperactivity disorder, autism Batten disease, Bechet's disease,blepharospasm, brain tumor, cerebral palsy Charcot-Marie-Tooth disease,chiari malformation, CIDP, non-Alzheimer-type dementia, dysautonomia,dyslexia, dysprazia, dystonia, epilepsy, essential tremor, Friedrich'sataxia, gaucher disease, Gullian-Barre syndrome, headache, migraine,Huntington's disease, hydrocephalus, Meniere's disease, motor neurondisease, multiple sclerosis, muscular dystrophy, myasthenia gravis,narcolepsy, Parkinson's disease, peripheral neuropathy, progressivesupranuclear palsy, restless legs syndrome, Rett syndrome,schizophrenia, Shy Drager syndrome, stroke, subarachnoid hemorrhage,Sydenham's syndrome, Tay-Sachs disease, Tourett syndrome, transientischemic attack, transverse myelitis, trigeminal neuralgia, tuberoussclerosis and von Hippel-Lindau syndrome.

In certain exemplary embodiments, Oligopaint kits are provided. As usedherein, the term “kit” refers to any delivery system for deliveringOligopaints and/or reagents for carrying out a method described herein.In the context of assays, such kits include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., an enclosureproviding one or more of, e.g., Oligopaints, primers (e.g., primersspecific for all Oligopaints present and/or one or more subsets ofprimers specific to one or more subsets of Oligopaint sequences) primershaving one or more detectable and/or retrievable labels bound thereto),supports having oligonucleotides bound thereto (e.g., microarrays,palettes, etc.), or the like) and/or supporting materials (e.g., anenclosure providing, e.g., buffers, written instructions for performingan assay described herein, or the like) from one location to another.For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materialsfor assays described herein. In one aspect, kits of the inventioncomprise Oligopaints specific for one or more target nucleotidesequences (e.g., chromosomes) or one or more regions of one or moretarget nucleotide sequences (e.g., sub-chromosomal regions). In anotheraspect, kits comprise one or more primer sequences, one or more supportshaving a plurality of synthetic, oligonucleotide sequences attachedthereto, and one or more detectable and/or retrievable labels. Suchcontents may be delivered to the intended recipient together orseparately. For example, a first container may contain primer sequencesfor use in an assay, while a second container may contain a supporthaving a plurality of synthetic, oligonucleotide sequences attachedthereto.

In certain embodiments, an Oligopaint kit provides one or more arraysand/or palettes having a plurality of specific oligonucleotide sequences(e.g., Oligopaints) bound thereto. In certain aspects, an array and/orpalette provides a plurality of oligonucleotide sequences (e.g.,Oligopaints) that is specific for a set of binding patterns in a genome(e.g., a human genome). In certain aspects, an array or palette isspecific for a set of chromosomal aberrations (e.g., one or more of atranslocation, an insertion, an inversion, a deletion, a duplication, atransposition, aneuploidy, polyploidy, complex rearrangement andtelomere loss) associated with one or more disorders described herein.In certain aspects, the Oligopaint kits described herein areparticularly suited for diagnostic and/or prognostic use for detectingone or more disorders described herein in clinical settings (e.g.,hospitals, medical clinics, medical offices, diagnostic laboratories,research laboratories and the like (e.g., for patient diagnosis and/orprognosis, prenatal diagnosis and/or prognosis and the like).

In certain aspects, an Oligopaint kit provides instructions foramplifying the plurality of specific oligonucleotide sequences (e.g.,Oligopaints) provided in the kit. In other aspects, the kit providesinstructions for detectably and/or retrievably labeling one or moretarget nucleic acid sequences (e.g., one or more chromosomes orsub-chromosomal regions) using the amplified Oligopaints. In otheraspects, an Oligopaint kit provides instructions for effectivelyremoving one or more of the plurality of specific oligonucleotidesequences (e.g., Oligopaints) during the amplification step by includingone or more unlabeled amplification primers that hybridizes to the oneor more oligonucleotide sequences that one wishes to remove, such thatthe one or more target nucleic acid sequences is rendered not detectablyand/or retrievably labeled.

In certain exemplary embodiments, a polynucleotide (e.g., an Oligopaint)has a retrievable label bound thereto. As used herein, the terms “bound”and “attached” refer to both covalent interactions and noncovalentinteractions. A covalent interaction is a chemical linkage between twoatoms or radicals formed by the sharing of a pair of electrons (i.e., asingle bond), two pairs of electrons (i.e., a double bond) or threepairs of electrons (i.e., a triple bond). Covalent interactions are alsoknown in the art as electron pair interactions or electron pair bonds.Noncovalent interactions include, but are not limited to, van der Waalsinteractions, hydrogen bonds, weak chemical bonds (i.e., via short-rangenoncovalent forces), hydrophobic interactions, ionic bonds and the like.A review of noncovalent interactions can be found in Alberts et al., inMolecular Biology of the Cell, 3d edition, Garland Publishing, 1994.

As used herein, the term “retrievable label” refers to a label that isattached to a polynucleotide (e.g., an Oligopaint) and can, optionally,be used to specifically and/or nonspecifically bind a target protein,peptide, DNA sequence, RNA sequence, carbohydrate or the like at or nearthe nucleotide sequence to which one or more Oligopaints havehybridized. In certain aspects, target proteins include, but are notlimited to, proteins that are involved with gene regulation such as,e.g., proteins associated with chromatin (See, e.g., Dejardin andKingston (2009) Cell 136:175), proteins that regulate (upregulate ordownregulate) methylation, proteins that regulate (upregulate ordownregulate) histone acetylation, proteins that regulate (upregulate ordownregulate) transcription, proteins that regulate (upregulate ordownregulate) post-transcriptional regulation, proteins that regulate(upregulate or downregulate) RNA transport, proteins that regulate(upregulate or downregulate) mRNA degradation, proteins that regulate(upregulate or downregulate) translation, proteins that regulate(upregulate or downregulate) post-translational modifications and thelike.

In certain aspects, a retrievable label is activatable. As used herein,the term “activatable” refers to a retrievable label that is inert(i.e., does not bind a target) until activated (e.g., by exposure of theactivatable, retrievable label to light, heat, one or more chemicalcompounds or the like). In other aspects, a retrievable label can bindone or more targets without the need for activation of the retrievablelabel.

In certain exemplary embodiments, a polynucleotide (e.g., an Oligopaint)has a detectable label bound thereto. As used herein, the term“detectable label” refers to a label that is attached to apolynucleotide (e.g., an Oligopaint) and can be used to identify atarget (e.g., a chromosome or a sub-chromosomal region) to which one ormore Oligopaints have hybridized. Typically, a detectable label isattached to the 3′- or 5′-end of a polynucleotide (e.g., an Oligopaint).Alternatively, a detectable label is attached to an internal portion ofan oligonucleotide (i.e., not at the 3′ or the 5′ end). Detectablelabels may vary widely in size and compositions; the followingreferences provide guidance for selecting oligonucleotide tagsappropriate for particular embodiments: Brenner, U.S. Pat. No.5,635,400; Brenner et al., Proc. Natl. Acad. Sci., 97: 1665; Shoemakeret al. (1996) Nature Genetics, 14:450; Morris et al., EP Patent Pub.0799897A1; Wallace, U.S. Pat. No. 5,981,179; and the like. In certainexemplary embodiments, a polynucleotide (e.g., an Oligopaint) includingone or more detectable labels can have a length within a range of from 4to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20nucleotides, respectively. In other exemplary embodiments apolynucleotide (e.g., an Oligopaint) including one or more detectablelabels can have a length of at least 30 nucleotides, at least 40nucleotides, at least 50 nucleotides, at least 60 nucleotides, at least70 nucleotides, at least 80 nucleotides, at least 90 nucleotides, atleast 100 nucleotides, at least 150 nucleotides, at least 200nucleotides, at least 300 nucleotides, at least 400 nucleotides, atleast 500 nucleotides, at least 600 nucleotides, at least 700nucleotides, at least 800 nucleotides, at least 900 nucleotides, atleast 1000 nucleotides or greater.

Methods for incorporating detectable labels into nucleic acid probes arewell known. Typically, detectable labels (e.g., as hapten- orfluorochrome-conjugated deoxyribonucleotides) are incorporated into anoligopaint during a polymerization or amplification step, e.g., by PCR,nick translation, random primer labeling, terminal transferase tailing(e.g., one or more labels can be added after cleavage of the primersequence), and others (see Ausubel et al., 1997, Current Protocols InMolecular Biology, Greene Publishing and Wiley-Interscience, New York).

In certain aspects, a suitable retrievable label or detectable labelincludes, but is not limited to, a capture moiety such as a hydrophobiccompound, an oligonucleotide, an antibody or fragment of an antibody, aprotein, a peptide, a chemical cross-linker, an intercalator, amolecular cage (e.g., within a cage or other structure, e.g., proteincages, fullerene cages, zeolite cages, photon cages, and the like), orone or more elements of a capture pair, e.g., biotin-avidin,biotin-streptavidin, NETS-ester and the like, a thioether linkage,static charge interactions, van der Waals forces and the like (See,e.g., Holtke et al., U.S. Pat. Nos. 5,344,757; 5,702,888; and U.S. Pat.No. 5,354,657; Huber et al., U.S. Pat. No. 5,198,537; Miyoshi, U.S. Pat.No. 4,849,336; Misiura and Gait, PCT publication WO 91/17160). Incertain aspects, a suitable retrievable label or detectable label is anenzyme (e.g., a methylase and/or a cleaving enzyme). In one aspect, anantibody specific against the enzyme can be used to retrieve or detectthe enzyme and accordingly, retrieve or detect an oligonucleotidesequence attached to the enzyme. In another aspect, an antibody specificagainst the enzyme can be used to retrieve or detect the enzyme and,after stringent washes, retrieve or detect an first oligonucleotidesequence that is hybridized to a second oligonucleotide sequence havingthe enzyme attached thereto.

Biotin, or a derivative thereof, may be used as an oligonucleotide(e.g., Oligopaint) label (e.g., as a retrievable label and/or adetectable label), and subsequently bound by a avidin/streptavidinderivative (e.g., detectably labeled, e.g., phycoerythrin-conjugatedstreptavidin), or an anti-biotin antibody (e.g., a detectably labeledantibody). Digoxigenin may be incorporated as a label and subsequentlybound by a detectably labeled anti-digoxigenin antibody (e.g., adetectably labeled antibody, e.g., fluoresceinated anti-digoxigenin). Anaminoallyl-dUTP residue may be incorporated into an oligonucleotide andsubsequently coupled to an N-hydroxy succinimide (NETS) derivatizedfluorescent dye, such as those listed infra. In general, any member of aconjugate pair may be incorporated into a retrievable label and/or adetectable label provided that a detectably labeled conjugate partnercan be bound to permit detection. As used herein, the term antibodyrefers to an antibody molecule of any class, or any sub-fragmentthereof, such as an Fab.

Other suitable labels (retrievable labels and/or detectable labels)include, but are not limited to, fluorescein (FAM), digoxigenin,dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU),hexahistidine (6×His), phosphor-amino acids (e.g. P-tyr, P-ser, P-thr)and the like. In one embodiment the following hapten/antibody pairs areused for retrieval and/or detection: biotin/α-biotin,digoxigenin/α-digoxigenin, dinitrophenol (DNP)/α-DNP,5-Carboxyfluorescein (FAM)/α-FAM.

Additional suitable labels (retrievable labels and/or detectable labels)include, but are not limited to, chemical cross-linking agents.Cross-linking agents typically contain at least two reactive groups thatare reactive towards numerous groups, including, but not limited to,sulfhydryls and amines, and create chemical covalent bonds between twoor more molecules. Functional groups that can be targeted withcross-linking agents include, but are not limited to, primary amines,carboxyls, sulfhydryls, carbohydrates and carboxylic acids. Proteinmolecules have many of these functional groups and therefore proteinsand peptides can be readily conjugated using cross-linking agents.Cross-linking agents are well known in the art and are commerciallyavailable (Thermo Scientific (Rockford, Ill.)).

Fluorescent labels and their attachment to oligonucleotides (e.g., toOligopaints) are described in many reviews, including Haugland, Handbookof Fluorescent Probes and Research Chemicals, Ninth Edition (MolecularProbes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition(Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991); Wetmur,Critical Reviews in Biochemistry and Molecular Biology, 26:227-259(1991); and the like. Particular methodologies applicable to theOligopaint methods and compositions described herein are disclosed inthe following sample of references: Fung et al., U.S. Pat. No.4,757,141; Hobbs, Jr., et al. U.S. Pat. No. 5,151,507; Cruickshank, U.S.Pat. No. 5,091,519. In one embodiment, one or more fluorescent dyes areused as labels for Oligopaints, e.g., as disclosed by Menchen et al.,U.S. Pat. No. 5,188,934 (4,7-dichlorofluorscein dyes); Begot et al.,U.S. Pat. No. 5,366,860 (spectrally resolvable rhodamine dyes); Lee etal., U.S. Pat. No. 5,847,162 (4,7-dichlororhodamine dyes); Khanna etal., U.S. Pat. No. 4,318,846 (ether-substituted fluorescein dyes); Leeet al., U.S. Pat. No. 5,800,996 (energy transfer dyes); Lee et al., U.S.Pat. No. 5,066,580 (xanthine dyes): Mathies et al., U.S. Pat. No.5,688,648 (energy transfer dyes); and the like. Labelling can also becarried out with quantum dots, as disclosed in the following patents andpatent publications: U.S. Pat. Nos. 6,322,901; 6,576,291; 6,423,551;6,251,303; 6,319,426; 6,426,513; 6,444,143; 5,990,479; 6,207,392;2002/0045045; 2003/0017264; and the like. Amines can be incorporatedinto Oligopaints, and labels can be added via the amines using methodsknown in the art. As used herein, the term “fluorescent label” includesa signaling moiety that conveys information through the fluorescentabsorption and/or emission properties of one or more molecules. Suchfluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer and the like.

Commercially available fluorescent nucleotide analogues readilyincorporated into the Oligopaints include, for example, Cy3-dCTP,Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, N.J.),fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXASRED™-5-dUTP,CASCADE BLUE™-7-dUTP, BODIPY TMFL-14-dUTP, BODIPY TMR-14-dUTP, BODIPYTMTR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXASRED™-12-dUTP, BODIPY™ 630/650-14-dUTP, BODIPY™ 650/665-14-dUTP, ALEXAFLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP,ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546-14-dUTP, fluorescein-12-UTP,tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADEBLUE™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP,RHODAMINE GREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, ALEXA FLUOR™ 546-14-UTP(Molecular Probes, Inc. Eugene, Oreg.). Protocols are available forcustom synthesis of nucleotides having other fluorophores. Henegariu etal., “Custom Fluorescent-Nucleotide Synthesis as an Alternative Methodfor Nucleic Acid Labeling,” Nature Biotechnol. 18:345-348 (2000).

Other fluorophores available for post-synthetic attachment include,inter alia, ALEXA FLUOR™ 350, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXAFLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493/503, BODIPYFL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650,BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissaminerhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, PacificBlue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, DYLIGHT™ DYES (e.g., DYLIGHT™ 405, DYLIGHT™ 488, DYLIGHT™549, DYLIGHT™ 594, DYLIGHT™ 633, DYLIGHT™ 649, DYLIGHT™ 680, DYLIGHT™750, DYLIGHT™ 800 and the like) (available from Thermo FisherScientific, Rockford, Ill.), Texas Red (available from Molecular Probes,Inc., Eugene, Oreg.), and Cy2, Cy3.5, Cy5.5, and Cy7 (available fromAmersham Biosciences, Piscataway, N.J. USA, and others).

FRET tandem fluorophores may also be used, such as PerCP-Cy5.5, PE-Cy5,PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7; also, PE-Alexa dyes (610,647, 680) and APC-Alexa dyes.

Metallic silver particles may be coated onto the surface of the array toenhance signal from fluorescently labeled oligonucleotide sequencesbound to an array. Lakowicz et al. (2003) BioTechniques 34:62.

Detection method(s) used will depend on the particular detectable labelsused in the Oligopaints. In certain exemplary embodiments, chromosomesand/or chromosomal regions having one or more Oligopaints bound theretomay be selected for and/or screened for using a microscope, aspectrophotometer, a tube luminometer or plate luminometer, x-ray film,a scintillator, a fluorescence activated cell sorting (FACS) apparatus,a microfluidics apparatus or the like.

When fluorescently labeled Oligopaints are used, fluorescencephotomicroscopy can be used to detect and record the results of in situhybridization using routine methods known in the art. Alternatively,digital (computer implemented) fluorescence microscopy withimage-processing capability may be used. Two well-known systems forimaging FISH of chromosomes having multiple colored labels bound theretoinclude multiplex-FISH (M-FISH) and spectral karyotyping (SKY). SeeSchrock et al. (1996) Science 273:494; Roberts et al. (1999) GenesChrom. Cancer 25:241; Fransz et al. (2002) Proc. Natl. Acad. Sci. USA99:14584; Bayani et al. (2004) Curr. Protocol. Cell Biol.22.5.1-22.5.25; Danilova et al. (2008) Chromosoma 117:345; U.S. Pat. No.6,066,459; and FISH TAG™ DNA Multicolor Kit instructions (Molecularprobes) for a review of methods for painting chromosomes and detectingpainted chromosomes.

In certain exemplary embodiments, images of fluorescently labeledchromosomes are detected and recorded using a computerized imagingsystem such as the Applied Imaging Corporation CytoVision System(Applied Imaging Corporation, Santa Clara, Calif.) with modifications(e.g., software, Chroma 84000 filter set, and an enhanced filter wheel).Other suitable systems include a computerized imaging system using acooled CCD camera (Photometrics, NU200 series equipped with Kodak KAF1400 CCD) coupled to a Zeiss Axiophot microscope, with images processedas described by Ried et al. (1992) Proc. Natl. Acad. Sci. USA 89:1388).Other suitable imaging and analysis systems are described by Schrock etal., supra; and Speicher et al., supra.

The in situ hybridization methods described herein can be performed on avariety of biological or clinical samples, in cells that are in any (orall) stage(s) of the cell cycle (e.g., mitosis, meiosis, interphase, G0,G1, S and/or G2). Examples include all types of cell culture, animal orplant tissue, peripheral blood lymphocytes, buccal smears, touchpreparations prepared from uncultured primary tumors, cancer cells, bonemarrow, cells obtained from biopsy or cells in bodily fluids (e.g.,blood, urine, sputum and the like), cells from amniotic fluid, cellsfrom maternal blood (e.g., fetal cells), cells from testis and ovary,and the like. Samples are prepared for assays of the invention usingconventional techniques, which typically depend on the source from whicha sample or specimen is taken. These examples are not to be construed aslimiting the sample types applicable to the methods and/or compositionsdescribed herein.

In certain exemplary embodiments, Oligopaints include multiplechromosome-specific probes, which are differentially labeled (i.e., atleast two of the chromosome-specific probes are differently labeled).Various approaches to multi-color chromosome painting have beendescribed in the art and can be adapted to the present inventionfollowing the guidance provided herein. Examples of such differentiallabeling (“multicolor FISH”) include those described by Schrock et al.(1996) Science 273:494, and Speicher et al. (1996) Nature Genet.12:368). Schrock et al. describes a spectral imaging method, in whichepifluorescence filter sets and computer software is used to detect anddiscriminate between multiple differently labeled DNA probes hybridizedsimultaneously to a target chromosome set. Speicher et al. describesusing different combinations of 5 fluorochromes to label each of thehuman chromosomes (or chromosome arms) in a 27-color FISH termed“combinatorial multifluor FISH”). Other suitable methods may also beused (see, e.g., Ried et al., 1992, Proc. Natl. Acad. Sci. USA89:1388-92).

Hybridization of the Oligopaints of the invention to target chromosomessequences can be accomplished by standard in situ hybridization (ISH)techniques (see, e.g., Gall and Pardue (1981) Meth. Enzymol. 21:470;Henderson (1982) Int. Review of Cytology 76:1). Generally, ISH comprisesthe following major steps: (1) fixation of the biological structure tobe analyzed (e.g., a chromosome spread), (2) pre-hybridization treatmentof the biological structure to increase accessibility of target DNA(e.g., denaturation with heat or alkali), (3) optional pre-hybridizationtreatment to reduce nonspecific binding (e.g., by blocking thehybridization capacity of repetitive sequences), (4) hybridization ofthe mixture of nucleic acids to the nucleic acid in the biologicalstructure or tissue; (5) post-hybridization washes to remove nucleicacid fragments not bound in the hybridization and (6) detection of thehybridized labelled oligonucleotides (e.g., hybridized Oligopaints). Thereagents used in each of these steps and their conditions of use varydepending on the particular situation. For instance, step 3 will notalways be necessary as the Oligopaints described herein can be designedto avoid repetitive sequences). Hybridization conditions are alsodescribed in U.S. Pat. No. 5,447,841. It will be appreciated thatnumerous variations of in situ hybridization protocols and conditionsare known and may be used in conjunction with the present invention bypractitioners following the guidance provided herein.

As used herein, the term “hybridization” refers to the process in whichtwo single-stranded polynucleotides bind non-covalently to form a stabledouble-stranded polynucleotide. The term “hybridization” may also referto triple-stranded hybridization. The resulting (usually)double-stranded polynucleotide is a “hybrid” or “duplex.” “Hybridizationconditions” will typically include salt concentrations of less thanabout 1 M, more usually less than about 500 mM and even more usuallyless than about 200 mM. Hybridization temperatures can be as low as 5°C., but are typically greater than 22° C., more typically greater thanabout 30° C., and often in excess of about 37° C. Hybridizations areusually performed under stringent conditions, i.e., conditions underwhich a probe will hybridize to its target subsequence. Stringentconditions are sequence-dependent and are different in differentcircumstances. Longer fragments may require higher hybridizationtemperatures for specific hybridization. As other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, the combination of parameters is more important thanthe absolute measure of any one alone. Generally, stringent conditionsare selected to be about 5° C. lower than the T_(m) for the specificsequence at s defined ionic strength and pH. Exemplary stringentconditions include salt concentration of at least 0.01 M to no more than1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3 and atemperature of at least 25° C. For example, conditions of 5×SSPE (750 mMNaCl, 50 mM Na phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C. are suitable for allele-specific probe hybridizations. For stringentconditions, see for example, Sambrook, Fritsche and Maniatis, MolecularCloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press (1989) andAnderson Nucleic Acid Hybridization, 1^(st) Ed., BIOS ScientificPublishers Limited (1999). “Hybridizing specifically to” or“specifically hybridizing to” or like expressions refer to the binding,duplexing, or hybridizing of a molecule substantially to or only to aparticular nucleotide sequence or sequences under stringent conditionswhen that sequence is present in a complex mixture (e.g., totalcellular) DNA or RNA.

In certain exemplary embodiments, synthesis of oligonucleotides (e.g.,Oligopaints) and/or amplification of oligonucleotides (e.g.,Oligopaints) can be performed using a support. In certain aspects,multiple supports (tens, hundreds, thousands or more) may be utilized(e.g., synthesized, amplified, hybridized or the like) in parallel.Suitable supports include, but are not limited to, slides (e.g.,microscope slides), beads, chips, particles, strands, gels, sheets,tubing (e.g., microfuge tubes, test tubes, cuvettes), spheres,containers, capillaries, microfibers, pads, slices, films, plates (e.g.,multi-well plates), microfluidic supports (e.g., microarray chips, flowchannel plates, biochips and the like) and the like. In variousembodiments, the solid supports may be biological, nonbiological,organic, inorganic or combinations thereof. When using supports that aresubstantially planar, the support may be physically separated intoregions, for example, with trenches, grooves, wells, or chemicalbarriers (e.g., lacking a lipid-binding coating). In exemplaryembodiments, supports can be made of a variety of materials including,but not limited to glass, quartz, ceramic, plastic, polystyrene,methylstyrene, acrylic polymers, titanium, latex, sepharose, cellulose,nylon and the like and any combination thereof. Such supports and theiruses are well known in the art.

In certain exemplary embodiments, supports may have functional groups ontheir surface which can be used to attach a lipid bilayer (e.g., aphospholipid bilayer) to the support. For example, at least a portion ofthe support can be coated with silane and dextran (e.g., high molecularweight dextran). Dextran in its hydrated form can function as amolecular cushion for the membrane and is capable of binding lipids onthe support. Suitable functional groups include, but are not limited to,silicon oxides (e.g., SiO₂), MgF₂, CaF₂, mica, polyacrylamide, dextranand the like and any combination thereof.

In certain exemplary embodiments, methods of generating and amplifyingsynthetic oligonucleotide sequences, e.g., Oligopaint sequences, areprovided. As used herein, the term “oligonucleotide” is intended toinclude, but is not limited to, a single-stranded DNA or RNA molecule,typically prepared by synthetic means. Nucleotides of the presentinvention will typically be the naturally-occurring nucleotides such asnucleotides derived from adenosine, guanosine, uridine, cytidine andthymidine. When oligonucleotides are referred to as “double-stranded,”it is understood by those of skill in the art that a pair ofoligonucleotides exists in a hydrogen-bonded, helical array typicallyassociated with, for example, DNA. In addition to the 100% complementaryform of double-stranded oligonucleotides, the term “double-stranded” asused herein is also meant to include those form which include suchstructural features as bulges and loops (see Stryer, Biochemistry, ThirdEd. (1988), incorporated herein by reference in its entirety for allpurposes). As used herein, the term “polynucleotide” is intended toinclude, but is not limited to, two or more oligonucleotides joinedtogether (e.g., by hybridization, ligation, polymerization and thelike).

The term “operably linked,” when describing the relationship between twonucleic acid regions, refers to a juxtaposition wherein the regions arein a relationship permitting them to function in their intended manner.For example, a control sequence “operably linked” to a coding sequenceis ligated in such a way that expression of the coding sequence isachieved under conditions compatible with the control sequences, such aswhen the appropriate molecules (e.g., inducers and polymerases) arebound to the control or regulatory sequence(s).

In certain exemplary embodiments, nucleotide analogs or derivatives willbe used, such as nucleosides or nucleotides having protecting groups oneither the base portion or sugar portion of the molecule, or havingattached or incorporated labels, or isosteric replacements which resultin monomers that behave in either a synthetic or physiologicalenvironment in a manner similar to the parent monomer. The nucleotidescan have a protecting group which is linked to, and masks, a reactivegroup on the nucleotide. A variety of protecting groups are useful inthe invention and can be selected depending on the synthesis techniquesemployed and are discussed further below. After the nucleotide isattached to the support or growing nucleic acid, the protecting groupcan be removed.

Oligonucleotides or fragments thereof may be purchased from commercialsources. Oligonucleotide sequences may be prepared by any suitablemethod, e.g., the phosphoramidite method described by Beaucage andCarruthers ((1981) Tetrahedron Lett. 22: 1859) or the triester methodaccording to Matteucci et al. (1981) J. Am. Chem. Soc. 103:3185), bothincorporated herein by reference in their entirety for all purposes, orby other chemical methods using either a commercial automatedoligonucleotide synthesizer or high-throughput, high-density arraymethods described herein and known in the art (see U.S. Pat. Nos.5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813,5,959,463, 4,861,571 and 4,659,774, incorporated herein by reference inits entirety for all purposes). Pre-synthesized oligonucleotides andchips containing oligonucleotides may also be obtained commercially froma variety of vendors.

In an exemplary embodiment, construction and/or selectionoligonucleotides may be synthesized on a solid support using masklessarray synthesizer (MAS). Maskless array synthesizers are described, forexample, in PCT application No. WO 99/42813 and in corresponding U.S.Pat. No. 6,375,903. Other examples are known of maskless instrumentswhich can fabricate a custom DNA microarray in which each of thefeatures in the array has a single stranded DNA molecule of desiredsequence. An exemplary type of instrument is the type shown in FIG. 5 ofU.S. Pat. No. 6,375,903, based on the use of reflective optics. It is adesirable that this type of maskless array synthesizer is under softwarecontrol. Since the entire process of microarray synthesis can beaccomplished in only a few hours, and since suitable software permitsthe desired DNA sequences to be altered at will, this class of devicemakes it possible to fabricate microarrays including DNA segments ofdifferent sequence every day or even multiple times per day on oneinstrument. The differences in DNA sequence of the DNA segments in themicroarray can also be slight or dramatic, it makes no difference to theprocess. The MAS instrument may be used in the form it would normally beused to make microarrays for hybridization experiments, but it may alsobe adapted to have features specifically adapted for the compositions,methods, and systems described herein. For example, it may be desirableto substitute a coherent light source, i.e., a laser, for the lightsource shown in FIG. 5 of the above-mentioned U.S. Pat. No. 6,375,903.If a laser is used as the light source, a beam expanded and scatterplate may be used after the laser to transform the narrow light beamfrom the laser into a broader light source to illuminate the micromirrorarrays used in the maskless array synthesizer. It is also envisionedthat changes may be made to the flow cell in which the microarray issynthesized. In particular, it is envisioned that the flow cell can becompartmentalized, with linear rows of array elements being in fluidcommunication with each other by a common fluid channel, but eachchannel being separated from adjacent channels associated withneighboring rows of array elements. During microarray synthesis, thechannels all receive the same fluids at the same time. After the DNAsegments are separated from the substrate, the channels serve to permitthe DNA segments from the row of array elements to congregate with eachother and begin to self-assemble by hybridization.

Other methods for synthesizing oligonucleotides (e.g., Oligopaints)include, for example, light-directed methods utilizing masks, flowchannel methods, spotting methods, pin-based methods, and methodsutilizing multiple supports.

Light directed methods utilizing masks (e.g., VLSIPS™ methods) for thesynthesis of oligonucleotides is described, for example, in U.S. Pat.Nos. 5,143,854, 5,510,270 and 5,527,681. These methods involveactivating predefined regions of a solid support and then contacting thesupport with a preselected monomer solution. Selected regions can beactivated by irradiation with a light source through a mask much in themanner of photolithography techniques used in integrated circuitfabrication. Other regions of the support remain inactive becauseillumination is blocked by the mask and they remain chemicallyprotected. Thus, a light pattern defines which regions of the supportreact with a given monomer. By repeatedly activating different sets ofpredefined regions and contacting different monomer solutions with thesupport, a diverse array of polymers is produced on the support. Othersteps, such as washing unreacted monomer solution from the support, canbe used as necessary. Other applicable methods include mechanicaltechniques such as those described in U.S. Pat. No. 5,384,261.

Additional methods applicable to synthesis and/or amplification ofoligonucleotides (e.g., Oligopaints) on a single support are described,for example, in U.S. Pat. No. 5,384,261. For example reagents may bedelivered to the support by either (1) flowing within a channel definedon predefined regions or (2) “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites.

Flow channel methods involve, for example, microfluidic systems tocontrol synthesis of oligonucleotides on a solid support. For example,diverse polymer sequences may be synthesized at selected regions of asolid support by forming flow channels on a surface of the supportthrough which appropriate reagents flow or in which appropriate reagentsare placed. One of skill in the art will recognize that there arealternative methods of forming channels or otherwise protecting aportion of the surface of the support. For example, a protective coatingsuch as a hydrophilic or hydrophobic coating (depending upon the natureof the solvent) is utilized over portions of the support to beprotected, sometimes in combination with materials that facilitatewetting by the reactant solution in other regions. In this manner, theflowing solutions are further prevented from passing outside of theirdesignated flow paths.

Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region. Typical dispensers include a micropipette to deliverthe monomer solution to the support and a robotic system to control theposition of the micropipette with respect to the support, or an ink-jetprinter. In other embodiments, the dispenser includes a series of tubes,a manifold, an array of pipettes, or the like so that various reagentscan be delivered to the reaction regions simultaneously.

Pin-based methods for synthesis of oligonucleotides on a solid supportare described, for example, in U.S. Pat. No. 5,288,514. Pin-basedmethods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-well microtitre dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In yet another embodiment, a plurality of oligonucleotides (e.g.,Oligopaints) may be synthesized on multiple supports. One example is abead based synthesis method which is described, for example, in U.S.Pat. Nos. 5,770,358, 5,639,603, and 5,541,061. For the synthesis ofmolecules such as oligonucleotides on beads, a large plurality of beadsare suspended in a suitable carrier (such as water) in a container. Thebeads are provided with optional spacer molecules having an active siteto which is complexed, optionally, a protecting group. At each step ofthe synthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

In certain embodiments, a plurality of oligonucleotides (e.g.,Oligopaints) may be synthesized, amplified and/or used in conjunctionwith beads and/or bead-based arrays. As used herein, the term “bead”refers to a discrete particle that may be spherical (e.g., microspheres)or have an irregular shape. Beads may be as small as approximately 0.1μm in diameter or as large approximately several millimeters indiameter. Beads typically range in size from approximately 0.1 μm to 200μm in diameter. Beads may comprise a variety of materials including, butnot limited to, paramagnetic materials, ceramic, plastic, glass,polystyrene, methylstyrene, acrylic polymers, titanium, latex,sepharose, cellulose, nylon and the like.

In certain aspects, beads may have functional groups on their surfacewhich can be used to oligonucleotides (e.g., Oligopaints) to the bead.Oligonucleotide sequences can be attached to a bead by hybridization(e.g., binding to a polymer), covalent attachment, magnetic attachment,affinity attachment and the like. For example, the bead can be coatedwith streptavidin and the nucleic acid sequence can include a biotinmoiety. The biotin is capable of binding streptavidin on the bead, thusattaching the nucleic acid sequence to the bead. Beads coated withstreptavidin, oligo-dT, and histidine tag binding substrate arecommercially available (Dynal Biotech, Brown Deer, Wis.). Beads may alsobe functionalized using, for example, solid-phase chemistries known inthe art, such as those for generating nucleic acid arrays, such ascarboxyl, amino, and hydroxyl groups, or functionalized siliconcompounds (see, for example, U.S. Pat. No. 5,919,523).

Various exemplary protecting groups useful for synthesis ofoligonucleotides on a solid support are described in, for example,Atherton et al., 1989, Solid Phase Peptide Synthesis, IRL Press. Invarious embodiments, the methods described herein utilize solid supportsfor immobilization of nucleic acids. For example, oligonucleotides maybe synthesized on one or more solid supports. Exemplary solid supportsinclude, for example, slides, beads, chips, particles, strands, gels,sheets, tubing, spheres, containers, capillaries, pads, slices, films,or plates. In various embodiments, the solid supports may be biological,nonbiological, organic, inorganic, or combinations thereof. When usingsupports that are substantially planar, the support may be physicallyseparated into regions, for example, with trenches, grooves, wells, orchemical barriers (e.g., hydrophobic coatings, etc.). Supports that aretransparent to light are useful when the assay involves opticaldetection (see e.g., U.S. Pat. No. 5,545,531). The surface of the solidsupport will typically contain reactive groups, such as carboxyl, amino,and hydroxyl or may be coated with functionalized silicon compounds (seee.g., U.S. Pat. No. 5,919,523).

In one embodiment, the oligonucleotides synthesized on the solid supportmay be used as a template for the production of Oligopaints. Forexample, the support bound oligonucleotides may be contacted withprimers that hybridize to the oligonucleotides under conditions thatpermit chain extension of the primers. The support bound duplexes maythen be denatured, pooled and subjected to further rounds ofamplification to produce Oligopaints in solution. In another embodiment,the support-bound oligonucleotides may be removed from the solid, pooledand amplified to produce Oligopaints in solution. The oligonucleotidesmay be removed from the solid support, for example, by exposure toconditions such as acid, base, oxidation, reduction, heat, light, metalion catalysis, displacement or elimination chemistry, or by enzymaticcleavage.

In one embodiment, oligonucleotides may be attached to a solid supportthrough a cleavable linkage moiety. For example, the solid support maybe functionalized to provide cleavable linkers for covalent attachmentto the oligonucleotides. The linker moiety may be one, two, three, four,five, six or more atoms in length. Alternatively, the cleavable moietymay be within an oligonucleotide and may be introduced during in situsynthesis. A broad variety of cleavable moieties are available in theart of solid phase and microarray oligonucleotide synthesis (see e.g.,Pon, R., Methods Mol. Biol. 20:465-496 (1993); Verma et al., Ann. Rev.Biochem. 67:99-134 (1998); U.S. Pat. Nos. 5,739,386, 5,700,642 and5,830,655; and U.S. Patent Publication Nos. 2003/0186226 and2004/0106728). A suitable cleavable moiety may be selected to becompatible with the nature of the protecting group of the nucleosidebases, the choice of solid support, and/or the mode of reagent delivery,among others. In an exemplary embodiment, the oligonucleotides cleavedfrom the solid support contain a free 3′-OH end. Alternatively, the free3′-OH end may also be obtained by chemical or enzymatic treatment,following the cleavage of oligonucleotides. The cleavable moiety may beremoved under conditions which do not degrade the oligonucleotides. Thelinker may be cleaved using two approaches, either (a) simultaneouslyunder the same conditions as the deprotection step or (b) subsequentlyutilizing a different condition or reagent for linker cleavage after thecompletion of the deprotection step.

The covalent immobilization site may either be at the 5′ end of theoligonucleotide or at the 3′ end of the oligonucleotide. In someinstances, the immobilization site may be within the oligonucleotide(i.e. at a site other than the 5′ or 3′ end of the oligonucleotide). Thecleavable site may be located along the oligonucleotide backbone, forexample, a modified 3′-5′ internucleotide linkage in place of one of thephosphodiester groups, such as ribose, dialkoxysilane, phosphorothioate,and phosphoramidate internucleotide linkage. The cleavableoligonucleotide analogs may also include a substituent on, orreplacement of, one of the bases or sugars, such as 7-deazaguanosine,5-methylcytosine, inosine, uridine, and the like.

In one embodiment, cleavable sites contained within the modifiedoligonucleotide may include chemically cleavable groups, such asdialkoxysilane, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, 5′-(N)phosphoramidate, and ribose. Synthesis andcleavage conditions of chemically cleavable oligonucleotides aredescribed in U.S. Pat. Nos. 5,700,642 and 5,830,655. For example,depending upon the choice of cleavable site to be introduced, either afunctionalized nucleoside or a modified nucleoside dimer may be firstprepared, and then selectively introduced into a growing oligonucleotidefragment during the course of oligonucleotide synthesis. Selectivecleavage of the dialkoxysilane may be effected by treatment withfluoride ion. Phosphorothioate internucleotide linkage may beselectively cleaved under mild oxidative conditions. Selective cleavageof the phosphoramidate bond may be carried out under mild acidconditions, such as 80% acetic acid. Selective cleavage of ribose may becarried out by treatment with dilute ammonium hydroxide.

In another embodiment, a non-cleavable hydroxyl linker may be convertedinto a cleavable linker by coupling a special phosphoramidite to thehydroxyl group prior to the phosphoramidite or H-phosphonateoligonucleotide synthesis as described in U.S. Patent ApplicationPublication No. 2003/0186226. The cleavage of the chemicalphosphorylation agent at the completion of the oligonucleotide synthesisyields an oligonucleotide bearing a phosphate group at the 3′ end. The3′-phosphate end may be converted to a 3′ hydroxyl end by a treatmentwith a chemical or an enzyme, such as alkaline phosphatase, which isroutinely carried out by those skilled in the art.

In another embodiment, the cleavable linking moiety may be a TOPS (twooligonucleotides per synthesis) linker (see e.g., PCT publication WO93/20092). For example, the TOPS phosphoramidite may be used to converta non-cleavable hydroxyl group on the solid support to a cleavablelinker. A preferred embodiment of TOPS reagents is the Universal TOPS™phosphoramidite. Conditions for Universal TOPS™ phosphoramiditepreparation, coupling and cleavage are detailed, for example, in Hardyet al, Nucleic Acids Research 22(15):2998-3004 (1994). The UniversalTOPS™ phosphoramidite yields a cyclic 3′ phosphate that may be removedunder basic conditions, such as the extended ammonia and/orammonia/methylamine treatment, resulting in the natural 3′ hydroxyoligonucleotide.

In another embodiment, a cleavable linking moiety may be an aminolinker. The resulting oligonucleotides bound to the linker via aphosphoramidite linkage may be cleaved with 80% acetic acid yielding a3′-phosphorylated oligonucleotide.

In another embodiment, the cleavable linking moiety may be aphotocleavable linker, such as an ortho-nitrobenzyl photocleavablelinker. Synthesis and cleavage conditions of photolabileoligonucleotides on solid supports are described, for example, inVenkatesan et al. J. of Org. Chem. 61:525-529 (1996), Kahl et al., J. ofOrg. Chem. 64:507-510 (1999), Kahl et al., J. of Org. Chem. 63:4870-4871(1998), Greenberg et al., J. of Org. Chem. 59:746-753 (1994), Holmes etal., J. of Org. Chem. 62:2370-2380 (1997), and U.S. Pat. No. 5,739,386.Ortho-nitrobenzyl-based linkers, such as hydroxymethyl, hydroxyethyl,and Fmoc-aminoethyl carboxylic acid linkers, may also be obtainedcommercially.

In another embodiment, oligonucleotides may be removed from a solidsupport by an enzyme such as a nuclease. For example, oligonucleotidesmay be removed from a solid support upon exposure to one or morerestriction endonucleases, including, for example, class IIs restrictionenzymes. A restriction endonuclease recognition sequence may beincorporated into the immobilized oligonucleotides and theoligonucleotides may be contacted with one or more restrictionendonucleases to remove the oligonucleotides from the support. Invarious embodiments, when using enzymatic cleavage to remove theoligonucleotides from the support, it may be desirable to contact thesingle stranded immobilized oligonucleotides with primers, polymeraseand dNTPs to form immobilized duplexes. The duplexes may then becontacted with the enzyme (e.g., a restriction endonuclease) to removethe duplexes from the surface of the support. Methods for synthesizing asecond strand on a support bound oligonucleotide and methods forenzymatic removal of support bound duplexes are described, for example,in U.S. Pat. No. 6,326,489. Alternatively, short oligonucleotides thatare complementary to the restriction endonuclease recognition and/orcleavage site (e.g., but are not complementary to the entire supportbound oligonucleotide) may be added to the support boundoligonucleotides under hybridization conditions to facilitate cleavageby a restriction endonuclease (see e.g., PCT Publication No. WO04/024886).

In yet another embodiment, a plurality of oligonucleotides (e.g.,Oligopaints) may be synthesized and/or amplified in solution. Methods ofsynthesizing oligonucleotide sequences are well-known in the art (See,e.g., Seliger (1993) Protocols for Oligonucleotides and Analogs:Synthesis and Properties, vol. 20, pp. 391-435, Efimov (2007)Nucleosides, Nucleotides & Nucleic Acids 26:8, McMinn et al. (1997) J.Org. Chem. 62:7074, Froehler et al. (1986) Nucleic Acids Res. 14:5399,Garegg (1986) Tet. Lett. 27:4051, Efimov (1983) Nucleic Acids Res.11:8369, Reese (1978) Tetrahedron 34:3143).

In certain embodiments, oligonucleotides (e.g., Oligopaints) are doublestranded (ds). In certain aspects, a ds oligonucleotide may besynthesized as two single stranded oligonucleotides that are hybridizedtogether, thus forming a ds oligonucleotide. Alternatively, a dsoligonucleotide may be synthesized is a ds form (e.g., using a ssoligonucleotide as a template). In other embodiments, oligonucleotides(e.g., Oligopaints) are single stranded (ss). In certain aspects, a ssoligonucleotide is generated in a ss form. In other aspects, a ssoligonucleotide is synthesized in a ds form and is converted to ss formsubsequent to synthesis using any of a variety of methods well known inthe art (e.g., by incorporating dUs into the ds oligonucleotide duringsynthesis that can be cleaved after synthesis, by chemical cleavageafter synthesis, by enzymatic cleavage after synthesis, by nucleasedigestion after synthesis, by light based cleavage after synthesis andthe like).

Exemplary chemically cleavable internucleotide linkages for use in themethods described herein include, for example, β-cyano ether,5′-deoxy-5′-aminocarbamate, 3′deoxy-3′-aminocarbamate, urea, 2′cyano-3′,5′-phosphodiester, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate,3′-(N)-phosphoramidate, 5′-(N)-phosphoramidate, a-amino amide, vicinaldiol, ribonucleoside insertion, 2′-amino-3′,5′-phosphodiester, allylicsulfoxide, ester, silyl ether, dithioacetal, 5′-thio-furmal,α-hydroxy-methyl-phosphonic bisamide, acetal, 3′-thio-furmal,methylphosphonate and phosphotriester. Internucleoside silyl groups suchas trialkylsilyl ether and dialkoxysilane are cleaved by treatment withfluoride ion. Base-cleavable sites include β-cyano ether,5′-deoxy-5′-aminocarbamate, 3′-deoxy-3′-aminocarbamate, urea,2′-cyano-3′, 5′-phosphodiester, 2′-amino-3′, 5′-phosphodiester, esterand ribose. Thio-containing internucleotide bonds such as3′-(S)-phosphorothioate and 5′-(S)-phosphorothioate are cleaved bytreatment with silver nitrate or mercuric chloride. Acid cleavable sitesinclude 3′-(N)-phosphoramidate, 5′-(N)-phosphoramidate, dithioacetal,acetal and phosphonic bisamide. An α-aminoamide internucleoside bond iscleavable by treatment with isothiocyanate, and titanium may be used tocleave a 2′-amino-3′,5′-phosphodiester-O-ortho-benzyl internucleosidebond. Vicinal diol linkages are cleavable by treatment with periodate.Thermally cleavable groups include allylic sulfoxide and cyclohexenewhile photo-labile linkages include nitrobenzylether and thymidinedimer. Methods synthesizing and cleaving nucleic acids containingchemically cleavable, thermally cleavable, and photo-labile groups aredescribed for example, in U.S. Pat. No. 5,700,642.

Enzymatic cleavage may be mediated by including a restrictionendonuclease cleavage site in the oligonucleotide sequence. Aftersynthesis of a ds oligonucleotide, the ds oligonucleotide may becontacted with one or more endonucleases to remove one strand. A widevariety of restriction endonucleases having specific binding and/orcleavage sites are commercially available, for example, from New EnglandBiolabs (Ipswich, Mass.).

In various embodiments, the methods disclosed herein compriseamplification of oligonucleotide sequences including, for example,Oligopaints. Amplification methods may comprise contacting a nucleicacid with one or more primers that specifically hybridize to the nucleicacid under conditions that facilitate hybridization and chain extension.Exemplary methods for amplifying nucleic acids include the polymerasechain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb.Symp. Quant. Biol. 51 Pt 1:263 and Cleary et al. (2004) Nature Methods1:241; and U.S. Pat. Nos. 4,683,195 and 4,683,202), anchor PCR, RACEPCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988)Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:360-364), self sustained sequence replication (Guatelli et al.(1990) Proc. Natl. Acad. Sci. U.S.A. 87:1874), transcriptionalamplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. U.S.A.86:1173), Q-Beta Replicase (Lizardi et al. (1988) BioTechnology 6:1197),recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; and Williamset al. (2002) J. Biol. Chem. 277:7790), the amplification methodsdescribed in U.S. Pat. Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797,6,124,090 and 5,612,199, or any other nucleic acid amplification methodusing techniques well known to those of skill in the art. In exemplaryembodiments, the methods disclosed herein utilize PCR amplification.

In certain exemplary embodiments, universal primers will be used toamplify nucleic acid sequences such as, for example, Oligopaints. Theterm “universal primers” refers to a set of primers (e.g., a forward andreverse primer) that may be used for chain extension/amplification of aplurality of polynucleotides, e.g., the primers hybridize to sites thatare common to a plurality of polynucleotides. For example, universalprimers may be used for amplification of all, or essentially all,polynucleotides in a single pool. In certain aspects, forward primersand reverse primers have the same sequence. In other aspects, thesequence of forward primers differs from the sequence of reverseprimers. In still other aspects, a plurality of universal primers areprovided, e.g., tens, hundreds, thousands or more.

In certain embodiments, the universal primers may be temporary primersthat may be removed after amplification via enzymatic or chemicalcleavage. In certain embodiments, the universal primers may be temporaryprimers that may be removed after amplification via enzymatic orchemical cleavage. In other embodiments, the universal primers maycomprise a modification that becomes incorporated into thepolynucleotide molecules upon chain extension. Exemplary modificationsinclude, for example, a 3′ or 5′ end cap, a label (e.g., fluorescein),or a tag (e.g., a tag that facilitates immobilization or isolation ofthe polynucleotide, such as, biotin, etc.).

In exemplary embodiments, primers may be designed to be temporary topermit removal of the primers. Temporary primers may be designed so asto be removable by chemical, thermal, light based, or enzymaticcleavage. Cleavage may occur upon addition of an external factor (e.g.,an enzyme, chemical, heat, light, etc.) or may occur automatically aftera certain time period (e.g., after n rounds of amplification). In oneembodiment, temporary primers may be removed by chemical cleavage. Forexample, primers having acid labile or base labile sites may be used foramplification. The amplified pool may then be exposed to acid or base toremove the primer at the desired location. Alternatively, the temporaryprimers may be removed by exposure to heat and/or light. For example,primers having heat labile or photolabile sites may be used foramplification. The amplified pool may then be exposed to heat and/orlight to remove the primer/primer binding sites at the desired location.In another embodiment, an RNA primer may be used for amplificationthereby forming short stretches of RNA/DNA hybrids at the ends of thenucleic acid molecule. The primer site may then be removed by exposureto an RNase (e.g., RNase H). In various embodiments, the method forremoving the primer may only cleave a single strand of the amplifiedduplex thereby leaving 3′ or 5′ overhangs. Such overhangs may be removedusing an exonuclease to form blunt ended double stranded duplexes. Forexample, RecJ_(f) may be used to remove single stranded 5′ overhangs andExonuclease I or Exonuclease T may be used to remove single stranded 3′overhangs. Additionally, S₁ nuclease, P₁ nuclease, mung bean nuclease,and CEL I nuclease, may be used to remove single stranded regions from anucleic acid molecule. RecJ_(f), Exonuclease I, Exonuclease T, and mungbean nuclease are commercially available, for example, from New EnglandBiolabs (Ipswich, Mass.). S1 nuclease, P1 nuclease and CEL I nucleaseare described, for example, in Vogt, V. M., Eur. J. Biochem., 33:192-200 (1973); Fujimoto et al., Agric. Biol. Chem. 38: 777-783 (1974);Vogt, V. M., Methods Enzymol. 65: 248-255 (1980); and Yang et al.,Biochemistry 39: 3533-3541 (2000).

In one embodiment, the temporary primers may be removed from a nucleicacid by chemical, thermal, or light based cleavage as described supra.In other embodiments, primers may be removed using enzymatic cleavage.For example, primers may be designed to include a restrictionendonuclease cleavage site. After amplification, the pool of nucleicacids may be contacted with one or more endonucleases to produce doublestranded breaks thereby removing the primers. In certain embodiments,the forward and reverse primers may be removed by the same or differentrestriction endonucleases. Any type of restriction endonuclease may beused to remove the primers/primer binding sites from nucleic acidsequences. In various embodiments, restriction endonucleases thatproduce 3′ overhangs, 5′ overhangs or blunt ends may be used.

In certain embodiments, it may be desirable to utilize a primercomprising one or more modifications such as a cap (e.g., to preventexonuclease cleavage), a linking moiety (such as those described aboveto facilitate immobilization of an oligonucleotide onto a substrate), ora label (e.g., to facilitate detection, isolation and/or immobilizationof a nucleic acid construct). Suitable modifications include, forexample, various enzymes, prosthetic groups, luminescent markers,bioluminescent markers, fluorescent markers (e.g., fluorescein),radiolabels (e.g., ³²P, ³⁵S, etc.), biotin, polypeptide epitopes, etc.as described further herein.

Embodiments of the present invention are directed to oligonucleotidesequences (e.g., Oligopaints) having one or more amplification sequencesor amplification sites. As used herein, the term “amplification site” isintended to include, but is not limited to, a nucleic acid sequencelocated at the 5′ and/or 3′ end of the oligonucleotide sequences of thepresent invention which hybridizes a complementary nucleic acidsequence. In one aspect of the invention, an amplification site isremoved from the oligonucleotide after amplification. In another aspectof the invention, an amplification site includes one or more restrictionendonuclease recognition sequences recognized by one or more restrictionenzymes. In another aspect, an amplification site is heat labile and/orphoto labile and is cleavable by heat or light, respectively. In yetanother aspect, an amplification site is a ribonucleic acid sequencecleavable by RNase. In still another aspect, an amplification site ischemically cleavable (e.g., using acid and/or base).

As used herein, the term “restriction endonuclease recognition site” isintended to include, but is not limited to, a particular nucleic acidsequence to which one or more restriction enzymes bind, resulting incleavage of a DNA molecule either at the restriction endonucleaserecognition sequence itself, or at a sequence distal to the restrictionendonuclease recognition sequence. Restriction enzymes include, but arenot limited to, type I enzymes, type II enzymes, type IIS enzymes, typeIII enzymes and type IV enzymes. The REBASE database provides acomprehensive database of information about restriction enzymes, DNAmethyltransferases and related proteins involved inrestriction-modification. It contains both published and unpublishedwork with information about restriction endonuclease recognition sitesand restriction endonuclease cleavage sites, isoschizomers, commercialavailability, crystal and sequence data (see Roberts et al. (2005) Nucl.Acids Res. 33:D230, incorporated herein by reference in its entirety forall purposes).

In certain aspects, primers of the present invention include one or morerestriction endonuclease recognition sites that enable type IIS enzymesto cleave the nucleic acid several base pairs 3′ to the restrictionendonuclease recognition sequence. As used herein, the term “type IIS”refers to a restriction enzyme that cuts at a site remote from itsrecognition sequence. Type IIS enzymes are known to cut at a distancesfrom their recognition sites ranging from 0 to 20 base pairs. Examplesof Type IIs endonucleases include, for example, enzymes that produce a3′ overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I,Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I,Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I,and Psr I; enzymes that produce a 5′ overhang such as, for example, BsmAI, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I,BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce ablunt end, such as, for example, Mly I and Btr I. Type-IIs endonucleasesare commercially available and are well known in the art (New EnglandBiolabs, Ipswich, Mass.). Information about the recognition sites, cutsites and conditions for digestion using type IIs endonucleases may befound, for example, on the Worldwide Web atneb.com/nebecomm/enzymefindersearch bytypeIIs.asp). Restrictionendonuclease sequences and restriction enzymes are well known in the artand restriction enzymes are commercially available (New EnglandBiolabs).

Certain exemplary embodiments are directed to the use of computersoftware to automate design and/or interpretation of genomic spacings,repeat-discriminating SNPs and/or colors for each specific oligopaintset. Such software may be used in conjunction with individualsperforming interpretation by hand or in a semi-automated fashion orcombined with an automated system. In at least some embodiments, thedesign and/or interpretation software is implemented in a programwritten in the JAVA programming language. The program may be compiledinto an executable that may then be run from a command prompt in theWINDOWS XP operating system. Unless specifically set forth in theclaims, the invention is not limited to implementation using a specificprogramming language, operating system environment or hardware platform.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, tables, andaccompanying claims.

Example I Overall Strategy for Oligopaint Design

1. Give centromeres an identifying color: e.g., can either make allcentromeres the same color, or make chromosome-specific.

2. Query whether minor M-bands will obscure major M-bands. Minor:majorM-band ratios such as, e.g., 1:1, 2, 3, 4, 5, 10, 20, 50, 100 will betried.

3. Query what distance the M-bands should be from one another to bedistinct. 250 and 500 kb, as well as 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10Mb will be tried.

4. Whether there is a pattern of I- and M-bands (varying color,thickness) that can uniquely identify regions will be determined. Allcombinations of 2, 3, 4, & 5 colors, spaced 50, 100, 200, 500, and 750kb, and 1 and 2 Mb apart for separation and color interference will betested. Five colors in a never-repeating pattern may permit unambiguoustagging of all genomic regions, but may raise challenges: a)condensation, which can also be uneven, may change colors. Varying bandwidths of a color may avoid issues because (without intending to bebound by scientific theory) condensation should not change hue.

5. How close (far apart) I-bands must be to make a single band (bedistinct) will be determined. Try 10, 30, 50, 75, 100, 150, 200, 500,750, and 1000 kb.

6. How many x-mers are needed to produce an I-band will be determined. Acontiguous 1, 3, 5, 7, and 10 kb will be labelled for all colors.

7. How long the probe/primer sequences should be will be determined.Probe lengths of 40-28 bases and corresponding primer lengths of 10-16bases will be tested, and this will be done for varying GC content.Pre-selections will be performed to avoid overlap with other primers andany unique sequence in the genome. Also, primers can be extended byadding tails after synthesis of array. Primers and extensions will bedesigned such that they do not overlap unique sequence other primers.

8. Watson & Crick strands will be separately labelled. Different primerswill be used for 5′ and 3′ ends, and N will be placed on only one of thetwo.

9. Whether universal primers should be used will be determined. Althoughuniversal primers can be used, although their usefulness is unclear.

10. Targets will be pre-selected to minimize partial homology torepetitive elements to avoid repetitive sequences. If problems ariseafter arrays are made, the following steps may be employed: a) competewith unlabeled probe, b) remove oligo from library by hybridizing w/ i)homologous RNA and removing by anti-RNA/DNA or ii) homologousDNA-biotin, putting through column.

11. Other avenues: a) Electron dense material will be used for EMstudies, b) bleeding of colors will be used to help study condensation.

12. Give unique colors to: ultraconserved elements (UCEs) (e.g.,intergenic/introic/exonic); imprinted regions; allelically skewed genes;exons; cell-type (e.g., stem) markers and the like.

Example II Restriction-Free Protocol to Make Oligopaint Probes

The basic idea is to make 60-mers on the Agilent platform: 10 base oneach end for the quasi-universal primers and 40 bases in the middlerepresenting the unique regions of the human genome (FIG. 5 ).

Parameters:

-   -   1. Minimum density 40-mer tiling (half that, ¼, ⅛, etc.)    -   2. Minimum length: 5 kb (3 kb, 7 kb, 9 kb)    -   3. Minimum interphase distance between bands: 40 kb (20, 60, 80)    -   4. Minimum metaphase distance between bands: 4 Mb (2, 6, 8)    -   5. Length of primers: 10 bp (9, 12, 14, 15)

Given a 40:1 compaction ratio for 30 nm chromatin, theDNA:Interphase(I):Metaphase(M) compaction ratios for chromosome 19 are(64 Mb) 21 mm:500 microns:5 microns=4000:100:1. A microscopic resolutionof 300 nm means 40 kbp/pixel interphase and 4 Mbp/pixel for metaphase.

Goals for Color Layout:

-   -   1) Know the location in I or M with minimal context and        counting.    -   2) Be suitable for human or computer reading    -   3) Have one set of paints to cover the interphase to metaphase        transition    -   4) Be able to uniformly label by whole chromosome or by arm or        by strand.    -   5) Be able to selectively amplify from up to 100 chips which        don't necessarily neatly end at the arm boundaries.

Assuming that 5 kb of solid (unique) 40-mers is enough to detect as aband in both I & M, 35 kb between bands in I, 4 Mb in M. Humanchromosomes range from 47 Mb=16 M-bands (#21) to 247 Mb=80 bands (#1).

Each set of 4 M-bands is enough to encode 5{circumflex over ( )}4=625bands (enough to cover the 800 such overlapping 16 Mbp regions with someredundancy considering that the I-bands contribute a 5th). Each M-bandhas 100 I-bands. Ten I-bands are enough to encode a unique (7 bit,2{circumflex over ( )}7=128) binary pattern, which can be augmented with3 check bits and repeated 10 times, for example:

Chromosome 1=4 sets of 4 M-bands—with I-band color in parenthesis, 50 to90 bands out of 100 (v. 1 out of 100 for M-bands): 4(5 . . . )4(5 . . .)4(5 . . . )4(5 . . . )1(5 . . . )4(5 . . . )4(5 . . . )4(5 . . . )2(see scenario #1 below).

Expanding the first of the 16 M-bands below:

-   -   Paint: 45555.55.5.5555.55.5.5555.55.5.5555.55.5.5555.55.5        .5555.55.5.45    -   1s digit 01234567890123456789012345678901234567890123456789        0123456789012    -   10s digit 00000000001111111111222222222233333333334444444444 . .        . 9999999999000    -   100s digit (Position 0 to 102)

A De Bruijn sequence B(k, n) is a cyclic sequence of a given alphabetsize k for which every possible subsequence of length n appears as asequence of consecutive characters exactly once (length=k{circumflexover ( )}n) (See Worldwide Websitehakank.org/comb/deBruijn.Applet.html).

Scenario #1: Chips are generated in order of position on the genome, soby labeling one chip out of N, that fraction of the genome is obtained(this is not going to perfectly coincide with a chromosome boundaryunless a few chips are wasted). Below are five B(4,4) sequences (5*256M-bands each) which should be more than enough for encoding the roughly800 M-bands (depending on optimal density from the first chipexperiment). In each set below the (missing) 5th color is the (dominant)I-band color. In this case each color has its own primer pair (5 total).Note that since these sequences are 256 characters long, they don't fiton the line but instead wrap to the next line. The first 256-mer belowassumes an I-band color #5:

-   -   444414442444344114412441344214422442344314432443341414241434111411241134        121412241234131413241334242434211421242134221422242234231423242334343114        312431343214322432343314332433311112111311221123113211331212131222122312        3212331313221323133213332222322332323333    -   555515552555355115512551355215522552355315532553351515251535111511251135        121512251235131513251335252535211521252135221522252235231523252335353115        312531353215322532353315332533311112111311221123113211331212131222122312        3212331313221323133213332222322332323333    -   444414442444544114412441544214422442544514452445541414241454111411241154        121412241254151415241554242454211421242154221422242254251425242554545114        512451545214522452545514552455511112111511221125115211551212151222122512        5212551515221525155215552222522552525555    -   444414445444344114415441344514455445344314435443341414541434111411541134        151415541534131413541334545434511451545134551455545534531453545334343114        315431343514355435343314335433311115111311551153113511331515131555155315        3515331313551353133513335555355335353333    -   444424445444344224425442344524455445344324435443342424542434222422542234        252425542534232423542334545434522452545234552455545534532453545334343224        325432343524355435343324335433322225222322552253223522332525232555255325        3525332323552353233523335555355335353333

Scenario #2: As per Scenario #1 except 24 I-band colors (A-X) are used,which means that the De Bruijn alphabet (for the M-bands) can only bek=3 (not 4 colors in Scenario #1) since now two colors are used just forthe I-bands. Below are 24 B(3,4) sequences (24*81M-bands each) whichshould be more than enough for encoding the roughly 800 M-bands(depending on optimal density from the first chip experiment). In eachset below the (missing) 1 or 2 colors combine to form the (dominant)I-band color (or 24 combinations total). Since each I-band has its oneprimer pair and the five primary colors have their own primer pairs (forthe M-bands), in principle anyone could get any combination ofchromosome and color combination and strand simply by how the primersare labeled (and independent of chip #). This can be easily extended toall 48 arms by assigning two primer pairs for each of the 24color-combinations (one each for p & q arms). Since these sequences are27 characters long, they don't fit on the line and instead wrap to thenext line. The first 27-mer below assumes an I-band color using #4 and 5or just #4 or just #5.

-   -   I:4&5: 33331333233113312332133223131323111311231213        1223232113212322132221111211221212222    -   I:3&5: 44441444244114412442144224141424111411241214        1224242114212422142221111211221212222    -   I:3&4: 55551555255115512552155225151525111511251215        1225252115212522152221111211221212222    -   I:2&5: 4 4 4 4 1 4 4 4 3 4 4 1 1 4 4 1 3 4 4 3 1 4 4 3 3 4 1 4 1        4 3 4 11 1 4 1 1 3 4 1 3 1 4 1 3 3 4 3 4 3 1 1 4 3 1 3 4 3 3 1 4        3 3 3 1 1 1 1 3 1 1 3 3 1 3 1 3 3 3 3    -   I:1&5: 4 4 4 4 2 4 4 4 3 4 4 2 2 4 4 2 3 4 4 3 2 4 4 3 3 4 2 4 2        4 3 4 2 2 2 4 2 2 3 4 2 3 2 4 2 3 3 4 3 4 3 2 2 4 3 2 3 4 3 3 2        4 3 3 3 2 2 2 2 3 2 2 3 3 2 3 2 3 3 3 3    -   I:1&4: 2 2 2 2 5 2 2 2 3 2 2 5 5 2 2 5 3 2 2 3 5 2 2 3 3 2 5 2 5        2 3 2 5 5 5 2 5 5 3 2 5 3 5 2 5 3 3 2 3 2 3 5 5 2 3 5 3 2 3 3 5        2 3 3 3 5 5 5 5 3 5 5 3 3 5 3 5 3 3 3 3    -   I:1&3: 2 2 2 2 5 2 2 2 4 2 2 5 5 2 2 5 4 2 2 4 5 2 2 4 4 2 5 2 5        2 4 2 5 5 5 2 5 5 4 2 5 4 5 2 5 4 4 2 4 2 4 5 5 2 4 5 4 2 4 4 5        2 4 4 4 5 5 5 5 4 5 5 4 4 5 4 5 4 4 4 4    -   I:1&2: 4 4 4 4 5 4 4 4 3 4 4 5 5 4 4 5 3 4 4 3 5 4 4 3 3 4 5 4 5        4 3 4 5 5 5 4 5 5 3 4 5 3 5 4 5 3 3 4 3 4 3 5 5 4 3 5 3 4 3 3 5        4 3 3 3 5 5 5 5 3 5 5 3 3 5 3 5 3 3 3 3    -   I:2&3: 4 4 4 4 5 4 4 4 1 4 4 5 5 4 4 5 1 4 4 1 5 4 4 1 1 4 5 4 5        4 1 4 5 5 5 4 5 5 1 4 5 1 5 4 5 1 1 4 1 4 1 5 5 4 1 5 1 4 1 1 5        4 11 1 5 5 5 5 1 5 5 1 1 5 1 5 11 1 1    -   I:2&4: 11 1 1 5 1 1 1 3 1 1 5 5 1 1 5 3 1 1 3 5 1 1 3 3 1 5 1 5        1 3 1 5 5 5 1 5 5 3 1 5 3 5 1 5 3 3 1 3 1 3 5 5 1 3 5 3 1 3 3 5        1 3 3 3 5 5 5 5 3 5 5 3 3 5 3 5 3 3 3 3

TABLE 1 Chromosome# Mb  1 447  4 444  4 400  4 191  5 181  6 171  7 159 8 146  9 140 10 145 11 144 14 144 14 114 14 106 15 100 16 89 17 79 1876 19 64 40 64 41 47 44 50 44-X 155 44-Y 58 4079

REFERENCES

-   Schrock et al. (1996) Science 474(5474):494-   Worldwide Website: ncbi.nlm.nih.gov/pubmed/11044455-   Worldwide Website: ncbi.nlm.nih.gov/pubmed/10479870-   Worldwide Website: ncbi.nlm.nih.gov/pubmed/8664547-   Cross-species color segmenting or RxFISH, barcodes from fragmented    hybrids (Worldwide Website: chrombios.com/AboutFISH/BarCodes.html)-   Multicolour (44 color) fluorescence in situ hybridisation (mFISH),    multicolour banding analysis (mBAND), region-specific partial    chromosome paints from Metasystems (Germany) (Worldwide Web site:    ori.nus.edu.es/MCytogenetics.html)-   Multicolor FICTION, DNA labelling were diethylaminocoumarin (DEAC),    SpectrumGreen™ (SG), SpectrumOrange™ (SO), Texas Red® (TR) and    Cyanine 5 (Cy™5), detection of the immunophenotype was performed    with aminomethylcoumarin (AMCA) (Worldwide Website:    metasystems.de/customers/a04/a04.htm)-   All STAR*FISH paint systems for whole human chromosomes (Worldwide    Website: openbiosystems.com/FISHprobes/Starfish/Human/Multicolor/)-   CTs 4 green (labeled with dinitrophenol, detected with FITC), CTs 5    blue (labeled with digoxigenin, detected with Cy4), and CTs 11 red    (labeled with biotin, detected with Cy5) (Worldwide Web site:    cshprotocols.cshlp.org/cgi/content/full/4007/10/pdb.prot4740/F4).

Example III Making Probes Using dU Digestion

To determine whether USER™-digested, synthesized oligonucleotides havingan internal fluor could be used in FISH, 60 base pair probes weresynthesized (as versus PCR amplified), mimicking what would be expectedif the oligonucleotides had been generated by PCR. The probes contained32 base pairs of homology to a locus in Drosophila that containsapproximately 110 copies of the target sequence. Both strands of a 60base pair oligonucleotide having internal dUs and internal fluors weresynthesized. The two synthesized oligonucleotides were mixed in equalportions and cleaved at the dUs with the USER™ (uracil-specific excisionreagent) enzyme (New England Biolabs, Ipswich, Mass.). Theoligonucleotides were then used for FISH, with a single-stranded 32 basepair oligonucleotide targeting a different sequence in the same regionas a control.

It was determined that double stranded, 32 base pair oligonucleotidescould be used as FISH probes, but double stranded, 60 base pairoligonucleotides could not. Since the double stranded PCR products wouldbe 60 base pairs in length, a strategy was developed for modifying themprior to FISH. PCR primers that carried an internal dU and an internalfluor were used such that the 5′ ends of the primers could be excisedwith USER™ subsequent to PCR (FIG. 7 ). It was determined thatUSER™-digested, synthesized (not PCR amplified) oligonucleotides couldbe used in FISH.

Having determined that the use of internal dUs and internal fluorspermitted synthesized, double stranded, 60 base pair oligonucleotides tobe used as probes, it was next queried whether analogous PCR generated60 base pair oligonucleotides could also be used as probes. It wasdetermined that USER™-digested, PCR generated, double stranded, 60 basepair oligonucleotides could indeed be used in FISH.

Synthesized Oligonucleotides

USER™-digested, synthesized oligonucleotides were used in FISH at 100ng, 200 ng, 400 ng and 800 ng concentrations. 200 ng of single stranded,32 base pair oligonucleotide was used as a control. FISH was performedas follows: 30 minute hybridization at room temperature, two 10 minutewashes, auto leveled using Photoshop, 60×objective, NA=1.2, 1 secondexposure.

PCR Generated Oligonucleotides

USER™-digested, PCR generated oligonucleotides were used in FISH at 50ng, 100 ng, 200 ng and 400 ng concentrations. 200 ng of single stranded,32 base pair oligonucleotide was used as a control. FISH was performedas follows: 30 minute hybridization at room temperature, two 10 minutewashes, auto leveled using Photoshop, 60×objective, NA=1.2, 1 secondexposure.

Example IV Enhancing Signal to Noise

Many protocols relying on hybridization of nucleic acid probes tonucleic acid targets aim to optimize signal to noise by increasing theaffinity of the probe to its target and decreasing the affinity of theprobe to background. The following strategies will be used to increasesignal to noise ratios:

1. The length of the probe will be extended via polymerization along anucleic acid target, e.g., a chromosome, thereby increasing the affinityof the probe to its target. Without intending to be bound by scientifictheory, probes that are incorrectly hybridized to targets ornon-specifically bound to non-nucleic acid substrates will not besubject to extension, thus increasing signal to noise ratios.

2. Probes that include one or more quenchers and one or more fluorescenttags will be used such that when a probe is hybridized to a nucleic acidtarget and extended, the quenchers will be released. Without intendingto be bound by scientific theory, this should enhance signal to noiseratios.

3. When hybridizing probes to cells or other complex targets, the amountof non-nucleic acid substrates present will be reduced through the useof proteinases, lipases and the like. Without intending to be bound byscientific theory, this should enhance signal to noise ratios.

Example V Oligopaints

Currently, companies such Open BioSystems and Metasystems useFACS-sorted chromosomes, which can also be microdissected into smallerfragments, to generate chromosome paints. This approach can provide upto 500 colored bands of per haploid genome (Metasystems), correspondingto approximately 6 Mb of DNA per band. The price of these paints rangesfrom approximately $100 to $4,000 per genome per assay, with chromosomepaints that provide higher resolution costing significantly more thanwhole chromosome paints.

The cost of paints can be greatly reduced by synthesizing them via PCRamplification of oligomers (e.g., 60-mers) that consist of genomicsequences (e.g., 32-mers) (representing only the unique part of thegenome) flanked by primer sequences (e.g., 14-mers) and, in total,represent 20% (although the oligomer lengths and percentages may differdepending on array optimization experiments, the type of genome, the ATcontent of the genome, spacing of repeated sequences with uniquesequences, etc.) of the human genome (FIG. 1 ). Oligomer sizes describedin this paragraph (e.g., primer sequences and/or genomic sequence) maybe increased or decreased based on the results of optimizationexperiments. These 60-mers will be synthesized on Agilent 244K arrays atthe cost of $500 per array such that 20% of the human genome will becontained on 80 to 95 arrays (FIG. 2 ). Judicious design and use of theprimer sequences will then, in conjunction with the subdivision of thegenome into 80 to 92 sub-chromosomal arrays, allow for the separateamplification and labeling of approximately 664 pools of genomicsequence. Application of all 664 pools of probe will then constitute awhole genome chromosome paint which, without intending to be bound byscientific theory, will produce a crisp banding pattern on metaphasechromosomes and increasingly finer banding patterns on increasinglydecondensed chromosomes (FIG. 3 ). Importantly, after the initialexpense of the arrays, the cost of maintaining the templates by PCR forthe future batches of paints will be minimal, dropping the cost of thepaints to dollars per assay (including the cost of primers and dyes).Each step of this protocol has been carried out successfully.

The Oligopaints and methods of making them described herein providenumerous advantages over chromosome paints that are commerciallyavailable. For example, Oligopaints and methods of making themprovide: 1) increased resolution over chromosome paints that arecommercially available; 2) reduced price over chromosome paints that arecommercially available; 3) availability for any organism for which thereis a genome sequence, even if that sequence is partial (that is, YACs,BACs and/or chromosomes that are sortable (e.g., by FACS) are notnecessary); 4) the ability to avoid background issues caused byrepetitive sequences, because the use of repetitive sequences can beavoided (in contrast, chromosome paints that are commercially availableuse “cold” (i.e., unlabeled) repeat sequences to outcompete the labeledprobes; 5) the option to eliminate certain bandings by not amplifyingprobes to those bands (e.g., after an array has been generated); 6) theoption to redesign arrays to fit individual needs; and 7) the ability tospecifically label certain sequences by giving them identifying primersequences.

The Oligopaints described herein are useful for a variety of methodsincluding, but not limited to: 1) heterologous and/or homologous (e.g.,pairing) interchromosomal interaction studies; 2) intrachromosomalorganizational studies such as, e.g., looping, coiling and the like; 3)chromosome organizational studies, such as, e.g., chromosome path,placement of specific sequences and the like; 4) chromosome condensationstudies, such as, e.g., mitosis, meiosis, arrest during cell cycle andthe like (new colors will be generated when Oligopainted bands overlapand/or ‘bleed’ into one another); 5) chromosome behavior studies suchas, e.g., segregation, motion in non-dividing cells and the like; 6)karyotyping studies such as, e.g., for medical science (e.g., diagnostickaryotyping, amniocentesis, pre-implantation diagnosis and the like),for basic science, to detect copy number variations (CNVs) and otherchromosomal rearrangements, changes in ploidy and the like; 7)replication studies such as, e.g., timing, organization, Bell nuclei andthe like; 8) chromosome structure studies such as, e.g., organization atthe electron microscopy level and the like; and 9) strand specificbiology of DNA through separate labeling of each of the two strands of aDNA double helix).

FIG. 1 illustrates how a new form of chromosome paints, Oligopaints, canbe made from template oligonucleotides which are synthesized on arrays(e.g., chips). Primers are annealed to sequences on the array and thenextended to generate a 60-mer products which could then be dissociatedfrom the array and used for second strand synthesis. Products are thenaliquoted into smaller pools which could be amplified with a singleprimer pair each. Finally, chromosome paints are made by amplifying thepools with primers containing two or more dU nucleotides and afluorescent dye at the 3′ end, followed by cleavage at the dUs to reduceinter-primer annealing. Note that if the two members of a primer pairare different in sequence, it will be possible to differentially labelthe two strands of DNA, enabling strand-specific hybridization to DNA orRNA. Note that the first few steps could be simplified if the originalsynthesized oligonucleotide can be released from the array. In certainexemplary embodiments, the use of dU allows for cleavage of primersequences (at the dU), which will reduce the concentration of primersequences present during hybridization.

FIG. 2 schematically depicts how the 24 chromosomes of the human genomecould be differentially colored with a base color for the interphasebands being a mix of five primary colors, and a series of color-codedmetaphase bands at staggered positions along the p and q arms. Thesepatterns will be generated by computer algorithms that select uniquesequences along the chromosomes and then associate them with primersequences such that their amplification with corresponding oligoscarrying the correct balance of dyes, followed by hybridization to thechromosome, which, without intending to be bound by scientific theory,will likely create identifying banding patterns for sub-chromosomalregions, especially when the chromosome is decondensed.

FIG. 1 shows how a band that appears thick on a metaphase chromosomewill likely disperse into a pattern of thinner bands, which it is hopedwill demarcate specific chromosomal regions on the order of 10-50 kb insize. Currently, arrays are being synthesized to a) confirm preliminarydata demonstrating that 14 base pair primers are sufficiently robust foruse in PCR amplification, b) determine how many oligonucleotide probesare necessary to generate a visible band in interphase and/or metaphase,c) determine how far apart oligonucleotide probes must be to generatedistinct bands in interphase and/or metaphase, d) assess what level ofvariation there may be in banding patterns from one chromosomal regionto another, e) determine what level of interference there may be whenfluorescent dyes are tightly packed, and f) ascertain whether theinterference can be taken advantage of to assess degrees of chromosomecondensation. Analogous arrays for the Drosophila and C. elegans genomesare also being designed for ongoing projects studying pairing in theseorganisms.

One innovative aspect of the oligopainting methods and compositionsdescribed herein is the use of computationally patterned syntheticprobes and arrays (rather than natural DNAs/chromosomes) to generatechromosome paints. This strategy will enable huge improvements in costand resolution.

Example VI Homology Effects

Oligopaint technology will enable the systematic investigation of tumorcells and cancer cell lines in terms of their chromosome arrangement andpositioning and, in doing so, will both emphasize experiments that areoften not routinely considered in terms of cancer as well as demonstratean affordable resource that will make such experiments generallyfeasible. Oligopaint technology will also enable the search for genesinvolved in somatic homolog pairing by permitting whole genomeFISH-based screens of the human genome using Oligopaints in the formatof 384-well plates. It was determined that FISH-based screens in384-well plates was successful. However, many in the art have predictedthat such an approach would not be technically and/or practicallyfeasible for whole genome screens, especially if the FISH were to targetunique sequences. Oligopaints will make this approach both technicallyand practically feasible. As a whole-genome Oligopaint FISH-basedstrategy offers a new for approach for identifying genes that affectchromosome organization, it will open up new lines of investigation. Inparticular, attempts will be made to identify genes that promote homologpairing, as such genes could be used to enhance gene replacement andgene therapy strategies that rely on homologous recombination.

Human chromosome karyotyping is a routine procedure for the analysis ofcancer genotypes as well as many genetic diseases, such those associatedwith a multitude of birth defects associated with whole chromosomeanueploidies, deletions, duplications, translocations and inversions.Furthermore, with the increased awareness of copy number variation andthe association of such chromosomal structures with disease, the demandfor karyotyping grows along with the need for increased accuracy.

Chromosome painting improves the power of karyotyping by color-codingchromosomes and sub-chromosomal segments. The ability of physicians tovisualize the underlying chromosomal basis for disease is key foraccurate diagnosis and treatment, making the availability of affordablepainting techniques a top priority in the medical innovation.

Better painting technologies will also impact the fields of geneticcounseling and prenatal diagnosis. Here, the accuracy of karyotyping isfrequently the determining factor in the quality of informationphysicians and genetics counselors can offer patient clients seekingexplanations for their ailments or wishing for a deeper understanding ofthe genotypes they have inherited and may pass on to their children.Unlike the karyotyping of patients whose disease syndromes will oftenhave already suggested likely chromosomal abnormalities, clients seekinggenetic counseling or prenatal diagnoses often seek information withoutany underlying syndromes. In these situations, the accuracy of theanalyses will rest to a great extent on the resolution of chromosomepaints across the entire genome and, therefore, the higher theresolution of the paints, the more reliable will be the informationobtained. Unfortunately, high resolution paints can cost thousands ofdollars for a single assay of an entire human genome. One goal is toproduce chromosome paints of the highest resolution for a fraction ofthe cost of current high resolution paints. As such, resources will beavailable to all populations, especially those for whom medical servicesare already an excessive financial burden.

Finally, the methods and compositions described in herein should affecta broad spectrum of research fields, including those focusing onchromosome organization, chromatin structure, interchromosomalinteractions, chromosome transmission, homology effects, replication,homologous recombination, genome integrity, and genome evolution. Thesefields center to a great extent on the concept of the chromosome as anentity in and of itself, something more than a repository of genes. Aschromosomes are difficult to study in their entirety except whenexamined in situ, protocols for visualizing them are of utmostimportance.

Using techniques ranging from traditional genetics to FISH, 3C analysis,4C analysis, 5C analysis and microarrays, researchers are cataloguinghundreds of interchromosomal interactions, some being specific betweentwo loci and others arising from the clustering of loci at transcriptionfactories or other nuclear structures. In short, the popular view of agene, with enhancers and promoters arrayed along a single black line,has been found lacking. The oligopainting methods and compositionsdescribed herein will enable one of skill in the art to focus onhomology effects. Homology effects encompass the many forms of generegulation that are sensitive to, or reflect, the presence of homologywithin a nucleus. The most celebrated of these would include threeprocesses that occur in humans and other mammals: X-inactivation, whereone of two X chromosomes is inactivated (Bacher et al. (2006) Science311:1149; Xu et al (2006) Science 311:1149, Epub 2006 Jan. 19))monoallelism (Borst (2002) Cell 109(1):5; Yang et al. (2007) Cell128:777), where only one allele of a gene is expressed, and parentalimprinting, a form of monoallelism that reflects the parental origin ofeach allele (Edwards et al. (2007) Curr. Opin. Cell Biol. 19:281; Pauleret al. (2007) Trends Genet. 23:284). Homology effects are also abundantin fungi, insects, worms, and plants. For example, a mere 450 base pairsof homology introduced by a transgene into the fungus, Neurospora, willtrigger C to T mutations within the duplicated regions (Selker (2004)Cold Spring Harb. Symp. Quant. Biol. 69:119), while 90 base pairs ofhomology between a transgene and the tobacco genome will causemethylation and silencing (Matzke and Matzke (2004) PLoS Biol. 2:E133).These phenomena demonstrate an uncanny ability of organisms to respondto homology and, as these responses to homology affect gene regulation,homology effects are of great relevance to human development and health.

Some homology effects are brought about through physical pairing of theinteracting homologous genes and/or chromosomal regions. Examples ofthese types of homology effects are now known to occur in a wide varietyof species, including humans and other mammals, insects and fungi. Amongthe most dramatic in mammals would be X-inactivation, where pairing ofthe X-inactivation center plays a role in the counting of X chromosomesand the subsequent process of inactivation. Mammals, like Neurospora,also sport a process call meiotic silencing of unpaired DNA/chromatin(MSUD/C) (Turner (2007) Development 134:1823), wherein regions of thegenome that remain unpaired in meiosis are silenced. Without intendingto be bound by scientific theory, this process may explain the curiousphenomenon of meiotic sex chromosome silencing, which occurs in malemeiosis and targets the unpaired regions of the X and Y chromosomes.

It has been determined that pairing can cause enhancers of a gene to actin trans on the promoter of another gene lying on a separate chromosome,and the cis-trans choice of an enhancer can be controlled by theintegrity of the promoter lying in cis to the enhancer. Pairing of aninternally deleted gene with a homolog bearing an insulator can lead tochanges in gene topology which allow bypass of the insulator (Morris etal. (1998) Proc. Natl. Acad. Sci. USA 95:10740). These two mechanisms ofpairing-mediated changes in gene regulation argue that somatic homologpairing is a potent form of gene regulation that warrants analysis inany diploid organism, including humans.

The oligopainting methods and compositions described herein can be usedto identify factors that mediate homolog pairing and, to this end,genetic screens have been conducted in Drosophila. While such anapproach has pointed to a handful of candidate genes involved in generegulation and chromosome structure (e.g., Hartl et al. (2008) Science322:1384; Williams et al. (2007) Genetics 177:31), progress has beenslow because past genetic screens have had to rely on observations ofpairing-sensitive phenotypes, which are sufficiently removed from theprocess of pairing that they can complicate analyses. These screens havealso been hindered by the need for organismal viability and themulti-tissue nature of the whole organism, which prohibits finer levelsof structural analyses. For these reasons, much effort has been exertedto establish a Drosophila cell culture system for the analysis ofpairing via FISH assays. Cell culture provides homogeneous populationsof cells for biochemical and molecular biological analyses (Ashe et al.(1997) Genes Dev. 11:2494).

A protocol permitting FISH assays in the 384-well format was developed.Using this protocol, sub-genome pilot runs surveyed 11% of the RNAilibrary representing the Drosophila genome, yielding a handful ofcandidate genes (FIG. 6 ). These runs addressed two important points.First, they documented the feasibility of FISH in the 384-well format.Second, they demonstrated the capacity of computerized image analysis todetect changes in the pattern of FISH signals from well to well. Asdiscussed further below, this protocol will be adapted for use withhuman cells.

The biology of pairing will be studied, and studies will begin with asurvey of human transformed cells taken directly from a wide variety oftumors as well as cell lines. FISH will be applied using whole genomeOligopaint methods and compounds described herein and to determine thestate of pairing along the length of each chromosome arm, takingadvantage of computer-based imaging techniques to allow the examinationof individual chromosomes or any combination of thereof.

To detect low levels of pairing or pairing that may be specific forcertain phases of the cell cycle, at least 100 cells per arm will bescored, and whether the cells appear to be entering mitosis or not willbe recorded. Because the degree of proximity may vary along a chromosomearm, measurements of inter-homolog distance will be made at multiplepositions along each arm, especially for the longer arms, by takingadvantage of bar coding implemented for Oligopaints.

The methods for measuring pairing will depend to a great extent on theresolution of the chromosome paints and whether and how well they willpermit the examination of decondensed chromosomes. Based in part on themethods and compositions described herein that provide Oligopaints at avery low cost, future experiments will not be limited in terms of probeand, therefore, a survey that is far-reaching and comprehensive will beable to be conducted.

Normal cells from a wide variety of tissue types will also be examined,as pairing has never been systematically assessed for humans or anymammal. Of the few studies looking for somatic pairing via FISH, probeshave generally targeted only single loci on single chromosome arms and,without intending to be bound by scientific theory, it remains possiblethat somatic pairing is more frequent than is currently predicted. Incontrast, studies will be performed with chromosome paints for all thechromosomes. Together with an analysis of transformed cells, thesestudies of normal cells will determine whether somatic pairing is acommon feature of human cells. Whether pairing is found only in renaloncocytomas (Koeman et al. (2008) PLoS Genetics 4:e1000176) or in othertransformed cells as well will be studied. If pairing is found outsideof renal oncocytomas, it will be studied whether it is restricted totransformed cells or whether it can occur in other types of diseasedtissues. Finally, if pairing can be found at a reasonable level in humancells, it will be studied whether it is restricted to only certainchromosomes. These are the important questions that can only be answeredby a broad, comprehensive, and unbiased sampling of cell types.

One advantage of whole genome chromosome painting over othertechnologies is that it will permit the analysis of interchromosomalinteractions at the single cell level. This resolution will addressquestions about cell-to-cell variation as well as correlations betweendifferent patterns of interchromosomal interactions that might beobscured when assays are done on large populations of cells. In short,although the methods and compositions described herein focus on pairing,Oligopaints will allow the analysis of other phenomena as well. Thesurvey of tumor and normal cells will be an important undertaking. Aspairing is a powerful modulator of gene expression, it is important,regardless of the outcome, to determine the level at which it occurs inhuman cells.

Experiments to identify genes in the human genome that are involved inhomolog pairing will be performed by conducting a whole-genomeRNAi-driven screen using FISH and chromosome paints to determine thestate of pairing on Chromosome 19. The pairing extends the entire lengthof the q arm of Chromosome 19, from centromere to telomere. Although theq arm is maximally paired, the p arm remains entirely unpaired. Further,pairing does not extend to any of the several other chromosomes thus farexamined. These three features of the pairing suggest an arm-based,rather than a locus-specific or whole-genome, mechanism for pairing.Without intending to be bound by scientific theory, one possibility forthese observations is that a Chromosome 19 q-arm-specific pairingmechanism has been induced in renal oncocytomas cell lines.Alternatively, without intending to be bound by scientific theory,pairing of the q arm in renal oncocytomas may result from the release ofa mechanism that normally suppresses pairing. Either way, it appearsthat pairing is a characteristic feature of renal oncocytomas broughtabout by a change that is inherited from cell-to-cell.

The gene or genes responsible for the pairing in renal oncocytomas willbe identified through a whole-genome RNAi-driven screen using FISH andchromosome paints as the phenotypic assay. Following a modified versionof the whole-genome, RNAi-driven, FISH-based protocol that was appliedto the Drosophila genome, cells will be grown in 384 well plates, andthey will be targeted with the repertoire of RNAi directed againstapproximately 20,000 human genes provided by the Institute for Chemistryand Cell Biology (ICCB) screening facility at Harvard Medical School.The impact of the RNAi will be assessed by visualizing the cells withFISH and chromosome paints targeting Chromosome 19, wherein the p and qarms will be differentially labeled. Using computer-aided analyses, RNAispecies that promote pairing of the q arm not the p arm will be searchedfor, although any pattern of pairing among the two arms will also be ofinterest. All candidates will be confirmed through additional runs ofRNAi, after which the genes identified by the most effective RNAispecies will be characterized through standard genetic, molecularbiological and biochemical studies for their role in chromosome pairingas well as tumorigenesis.

The cost of the probe for the screen described above will cost onlyapproximately $1,200 using the Oligopaint methods and compositionsdescribed herein for Chromosome 19. This would be in sharp contrast tocosts of between $90,000 (Metasystems) and $700,000 (Open Biosystems) oras much as $1,400,000 (Metasystems), if the resolution of paints to bepurchased were to match that of the Oligopaints that will be synthesizedfor Chromosome 19.

The screen can also be carried out with Oligopaints to the entire genomeso that the impact of RNAi species on the pairing of all chromosome armscan be displayed simultaneously. Oligopaint costs for such a globalscreen would be approximately $28,000. This global approach may beattempted or, alternatively, an approach that simultaneously targetsseveral, but not all, of the chromosomes may be undertaken. Along theselines, a few technical modifications may be necessary in the adaptationof the Drosophila screening protocol to the protocol above. Inparticular, the Drosophila screen used short oligonucleotide probescontaining locked nucleic acids (LNAs), which allowed a more facileadaptation of the FISH protocol to the 384-well plate format. Inaddition, or alternatively, Oligopaints may incorporate LNAs. If thisroute is taken, it may reduce the ‘density’ of probes in the paints inorder to offset the greater cost of LNAs as compared to that ofunmodified bases. In the unlikely case that neither of these approachesis successful, a screen of the human genome will be performed using afew LNA probes along Chromosome 19, following, exactly, the protocolthat was used for screening the Drosophila genome.

REFERENCES

-   Koeman J M, Russell R C, Tan M H, Petillo D, Westphal M, Furge K A.    Somatic pairing of chromosome 19 in renal oncocytoma is associated    with deregulated EGLN2-mediated [corrected] oxygen-sensing response    PLoS Genet. 2008 July; 4(7)-   Osborne C S, Chakalova L, Mitchell J A, Horton A, Wood A L, Bolland    D J, Corcoran A E, Fraser P. Myc dynamically and preferentially    relocates to a transcription factory occupied by Igh. PLoS Biol.    2007 August; 5(8):e192.-   Cooper G M, Nickerson D A, Eichler E E. Mutational and selective    effects on copy-number variants in the human genome. Nat Genet. 2007    July; 39(7 Suppl):522-9.-   Matsuda K, Tanaka M, Araki S, Yanagisawa R, Yamauchi K, Koike K.    Cryptic insertion into 11q23 of MLLT10 not involved in    t(1;15;11;10)(p36;q11;q23;q24) in infant acute biphenotypic    leukemia. Cancer Genet. Cytogenet. 2009 Apr. 15; 190(2): 113-20-   Spilianakis, C. G., M. D. Lalioti, T. Town, G. R. Lee and R. A.    Flavell, 2005 Interchromosomal associations between alternatively    expressed loci. Nature 435: 637-645.-   Ling, J. Q., T. Li, J. F. Hu, T. H. Vu, H. L. Chen et al., 2006 CTCF    mediates interchromosomal colocalization between Igf2/H19 and    Wsb1/Nf1. Science 312: 269-272.-   Bacher C P, Guggiari M, Brors B, Augui S, Clerc P, Avner P, Eils R,    Heard E. 2006. Transient colocalization of X-inactivation centres    accompanies the initiation of X inactivation. Nat. Cell Biol.    8:293-9. Epub 2006 Jan. 24.-   Xu N, Tsai C L, Lee J T. 2006. Transient homologous chromosome    pairing marks the onset of X inactivation. Science. 311:1149-52.    Epub 2006 Jan. 19.

What is claimed is:
 1. A method comprising: contacting a plurality ofoligopaints to a target nucleic acid sequence, wherein contactedoligopaints form a barcode or combinatorial color sequence that enablesreading of a high number of target nucleic acid sequences.
 2. The methodof claim 1, wherein the target nucleic acid sequence comprises a DNAsequence or an RNA sequence.
 3. The method of claim 1, wherein thetarget nucleic acid sequence is an oligonucleotide.
 4. The method ofclaim 1, wherein the target nucleic acid sequence is linked to aprotein.
 5. The method of claim 4, wherein the protein comprises anantibody.
 6. The method of claim 1, wherein the contactedoligonucleotide paints are in contact with a retrievable label.
 7. Themethod of claim 1, wherein the oligonucleotide paint is configured to beidentifiable with a retrievable label.
 8. The method of claim 7, whereinthe retrievable label is configured to bind a moiety selected from thegroup consisting of a protein, a peptide, a DNA sequence, an RNAsequence, and a carbohydrate.
 9. The method of claim 7, wherein theprotein, the peptide, the DNA sequence, the RNA sequence, and thecarbohydrate are in a cell.
 10. The method of claim 1, wherein the highnumber of target nucleic sequences comprises at least one of 50 or moretargets, 100 or more targets, 1000 or more targets, 10,000 or moretargets.
 11. The method of claim 1, wherein the high number of targetnucleic acid sequences comprises a number of targets that encode 1-99%of a genome.
 12. The method of claim 11, wherein the genome comprises ahuman genome.
 13. The method of claim 12, wherein detecting occurs in acell, and the method further comprising karyotyping the cell.
 14. Themethod of claim 12, wherein detecting occurs in a cell, and the methodfurther comprises detecting a chromosomal aberration in a cell.
 15. Themethod of claim 1, wherein the contacting occurs within a cell.
 16. Themethod of claim 15, wherein the cell is a human cell.
 17. The method ofclaim 1, wherein detecting occurs in a biological tissue.
 18. The methodof claim 17, wherein the biological tissue is human tissue.